pumilio: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - pumilio

Synonyms -

Cytological map position - 85C-D

Function - mRNA binds to 3'UTR of Hunchback mRNA

Keyword(s) - posterior group

Symbol - pum

FlyBase ID:FBgn0003165

Genetic map position - 3-48.5

Classification - novel RNA binding protein

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Burow, D. A., Umeh-Garcia, M. C., True, M. B., Bakhaj, C. D., Ardell, D. H. and Cleary, M. D. (2015). Dynamic regulation of mRNA decay during neural development. Neural Dev 10: 11. PubMed ID: 25896902
Summary:
Gene expression patterns are determined by rates of mRNA transcription and decay. While transcription is known to regulate many developmental processes, the role of mRNA decay is less extensively defined. A critical step toward defining the role of mRNA decay in neural development is to measure genome-wide mRNA decay rates in neural tissue. This study developed a technique that allows genome-wide mRNA decay measurements in intact Drosophila embryos, across all tissues and specifically in the nervous system. The approach revealed neural-specific decay kinetics, including stabilization of transcripts encoding regulators of axonogenesis and destabilization of transcripts encoding ribosomal proteins and histones. Correlations were identified between mRNA stability and physiologic properties of mRNAs; mRNAs that are predicted to be translated within axon growth cones or dendrites have long half-lives while mRNAs encoding transcription factors that regulate neurogenesis have short half-lives. A search for candidate cis-regulatory elements identified enrichment of the Pumilio recognition element (PRE) in mRNAs encoding regulators of neurogenesis. Decreased expression of the RNA-binding protein Pumilio was found to stabilized predicted neural mRNA targets, and a PRE was found to be necessary to trigger reporter-transcript decay in the nervous system. In conclusion it was found that differential mRNA decay contributes to the relative abundance of transcripts involved in cell-fate decisions, axonogenesis, and other critical events during Drosophila neural development. Neural-specific decay kinetics and the functional specificity of mRNA decay suggest the existence of a dynamic neurodevelopmental mRNA decay network. Pumilio was found to be one component of this network, revealing a novel function for this RNA-binding protein.
Weidmann, C.A., Qiu, C., Arvola, R.M., Lou, T.F., Killingsworth, J., Campbell, Z.T., Tanaka Hall, T.M. and Goldstrohm, A.C. (2016). Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio. Elife [Epub ahead of print]. PubMed ID: 27482653
Summary:
Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet understanding of these key regulatory complexes has been limited to single RBPs. This study investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. The obtained results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. These results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.
Lin, W. H., Giachello, C. N. and Baines, R. A. (2016). Seizure control through genetic and pharmacological manipulation of Pumilio: a key component of neuronal homeostasis. Dis Model Mech [Epub ahead of print]. PubMed ID: 28067623
Summary:
Epilepsy is a significant disorder for which approximately one-third of patients do not respond to drug treatments. Next-generation drugs, which interact with novel targets, are required to provide a better clinical outcome for these individuals. To identify potential novel targets for antiepileptic drug (AED) design, this study used RNA sequencing to identify changes in gene transcription in two seizure models of the fruitfly Drosophila melanogaster. The first model compared gene transcription between wildtype (WT) and the bangsenseless1 (parabss) mutant; a gain-of-function in the sole fly voltage-gated sodium channel (paralytic). The second model compared WT to WT fed the proconvulsant picrotoxin (PTX). 743 genes with significant altered expression levels were identified that are common to both seizure models. Of these, 339 are up-regulated and 397 are down-regulated. pumilio (pum) was down-regulated in both seizure models. Pum is a known homeostatic regulator of action potential firing in both flies and mammals. Pum achieves control of neuronal firing through binding to, and regulating translation of, the mRNA transcripts of voltage-gated sodium channels (Nav). Maintaining expression of pum in the CNS of parabss is potently anticonvulsive, whilst its reduction through RNAi-mediated knockdown is proconvulsive. Using a cell-based luciferase reporter screen, a repurposed chemical library was screened, and 12 compounds sufficient to increase activity of dPum were identified. Of these compounds, this study focused on avobenzone which significantly rescues seizure behaviour in parabss. The mode-of-action of avobenzone includes potentiation of pum expression and mirrors the ability of this homeostatic regulator to reduce the persistent voltage-gated Na+ current (INaP) in an identified neuron. This study reports a novel approach to suppress seizure and highlights the mechanisms of neuronal homeostasis as potential targets for next-generation AEDs.
BIOLOGICAL OVERVIEW

pumilio is a posterior group gene: genes in this group are considered essential for the development of the posterior of the fly. The protein is supplied by the mother, and is ready to go to work even before fertilization takes place. A discussion of the PUM protein is clarified by looking at what takes place just before the protein functions. For PUM, this requires an examination of Nanos. Nanos governs the posterior patterning of Drosophila embryos. Localization of NOS mRNA assures that NOS protein is present in the posterior and can thus carry out inhibition of translation of Hunchback mRNA, thus allowing for the development of posterior fate.

Experiments have suggested that NOS mRNA is properly localized in pum- mutants, indicating that pum must function after the localization of NOS mRNA (Barker, 1992). It has been assumed that NOS itself is sufficient for inhibition of hunchback translation. The recent observation that PUM protein binds specifically to the Nanos response elements (NRE) of HB mRNA now clarifies the roles of NOS and PUM proteins in repression of HB mRNA translation. PUM protein is recruited first to HB mRNA, followed presumbly by NOS, recruited via protein-protein interactions. The two proteins together act as translational inhibitors (Murata, 1995).

Pumilio has an earlier role in regulation of asymmetric division of germline stem cells in the Drosophila ovary. pumilio mutations are known to affect the asymmetric division of germline stem cells in the Drosophila ovary. Germline stem cells play a pivotal role in gametogenesis; yet little is known about how they are formed, how they divide to self-renew, and how these processes are genetically controlled. Self-renewing asymmetric division of germline stem cells takes place in the Drosophila ovarian germline, as marked by the spectrosome, a cytoplasmic structure rich in membrane skeletal proteins including Spectrin. In the Drosophila ovary, germline stem cells, whose progeny ultimately give rise to eggs, are among the 3 to 5 most apically located germ cells in the germarium -- they are located towards the narrow end of the ovary, at the opposite end from the mature eggs. Terminal filament cells, somatic cells that make up the most proximal part of the ovary close to the stem cells, stain strongly for anti-alpha spectin antibody. The base of the terminal filament contains two to three squamous somatic cells rather than a single basal cell. The basal terminal filament cells retain strong anti-spectrin staining. These basal cells are in contact with two to three underlying germ cells. In these germ cells the spectrosome is usually apically located in the cytoplasm, closely apposed to the basal terminal filament cells. Occasionally, the spectrosome is not in contact with the basal cells. Even so, it is still tethered to the basal cell by a thin filament (Lin, 1997).

The ontogeny of the spectrosome marks the lineage of germline stem cells. The 2-3 most apical germ cells contacting the basal terminal filament cells show striking asymmetry in two ways with regard to the behavior of the spectrosome during mitosis: (1) one pole of their mitotic spindles is always associated with the spectrosome and the terminal filament, clearly marking a cytological asymmetry of the division. (2) The mitotic spindles are oriented along the apico-basal axis of the germarium generating a daughter cell in contact with the basal cell and another daughter cell that is one cell away from the basal cell. This distal daughter cell undergoes incomplete divisions to form germline cysts that ultimately give rise to nurse cells and the egg. Mutations have been identified in which the divisional asymmetry is disrupted. One set of mutations, referred to as ovarette (ovt) mutations, corresponds to a novel class of mutations in the pumilio locus (Lin, 1997).

pumilio mutation produces a small ovary phenotype. To examine whether this phenotype is the result of stem cell dysfunction, the germaria of females bearing a strong ovt mutation were examined. None of the mutant germ cells in pum mutant germaria appear to undergo asymmetric divisions. Instead, they contain only two or three clusters of apparently undifferentiated germline cells that do not contain a spectrosome. This defect suggests that the 2-3 mutant germline stem cells have undergone symmetric divisions to produce clusters of undifferentiated germline cells. Interestingly, lack of a spectrosome does not need to affect the rate of stem cell division. Mutation in the gene hu li tai shao (hts) coding for the cytoskeletal protein adducin, abolishes the spectrosome but does not affect the rate of stem cell division. In hts mutant females lacking a spectrosome, cysts continue to be produced at essentially normal rates. However, hts cystoblasts undergo a drastically modified process of cyst formation and are unable to differentiate an oocyte, presumably due to their inability to support a spectrosome derived fusome (Lin, 1997).

Since pumilio is known to posttranscriptionally repress the expression of Nanos at the earliest stages of germ cell development (Kobayashi, 1996), these results suggest that a similar activity is needed to maintain germ line stem cells. The simplest interpretation of the role of Pumilio in stem cell dynamics is that Pum, and likely Nos as well, act in the stem cell itself where they might act in concert with germ cell-specific molecules, such as Vasa, to participate in translational suppression, which keeps certain genes inactive until specific times in development or in the cell cycle. Inappropriate expression of the suppressed genes would cause the stem cells to assume a cystoblast identity or to proceed down an abnormal developmental pathway leading to the undifferentiated cell clusters (Lin, 1997).

Besides playing a role in ovaries in germ cell development, Pum also acts during embryogenesis to regulate germline development. The maternal RNA-binding proteins Pumilio (Pum) and Nanos (Nos) accumulate in pole cells, the germline progenitors. Nos is required for pole cells to differentiate into functional germline. Pum is also essential for germline development in embryos. A mutation in pum causes a defect in pole-cell migration into the gonads. In such pole cells, the expression of a germline-specific marker (PZ198) is initiated prematurely. pum mutation causes premature mitosis in the migrating pole cells. Pum inhibits pole-cell division by repressing translation of cyclin B messenger RNA. Because these phenotypes are indistinguishable from those produced by nos mutation, it is concluded that Pum acts together with Nos to regulate these germline-specific events (Asaoka-Taguchi, 1999).

Pole cells formed in embryos lacking Pum (pum embryos) were transplanted into wild-type host embryos. The transplanted pum pole cells pass normally through the midgut epithelium into the haemocoel. However, none of the transplanted pum pole cells are incorporated within the gonads of the hosts, whereas normal pole cells taken from control embryos were observed in the gonads. All of the transplanted pum pole cells remain in the haemocoel and the gut lumen. These results show that Pum is autonomously required in pole cells for their migration into the gonads. The expression of the enhancer-trap marker PZ198 was studied in pum pole cells. PZ198 expression, which is normally initiated in pole cells within the gonads, begins prematurely during pole-cell migration in embryos lacking Nos (nos embryos). Similarly, PZ198 expression begins prematurely, at stage 7, in pum mutant pole cells, as compared with stage 13 in control embryos. Thus Pum is also required to repress the premature expression of the enhancer-trap marker in pole cells. The effects of pum and nos mutations on cell-cycle arrest were studied during pole-cell migration. In normal development, pole cells remain quiescent in G2 phase of the cell cycle during stages 7-15. It is expected that Nos and Pum repress the entry of pole cells into mitosis, because pole cells initiate cell division just after Nos becomes undetectable in pole cells at stage 15. To monitor the cell cycle in pole cells, antibodies against a phosphorylated form of histone H3 (PH3) and cyclin E were used. PH3 is detectable in mitosis but is absent during interphase, whereas cyclin E is expressed specifically in S and G2 phases. The disappearance of cyclin E from pole cells is linked to cell-cycle progression from G2 to G1 phase, whereas cyclin E is not degraded during cell cycling of somatic cells. Consistent with the observation that migrating pole cells in wild-type embryos are arrested in G2 phase, almost all pole cells in stage 7-15 embryos show cyclin E staining, but not PH3 staining. In contrast, in pum and nos embryos, the percentage of pole cells expressing cyclin E gradually decreases during stages 7-15, and PH3-positive pole cells became detectable during these stages. Thus, the mutant pole cells are prematurely released from G2 arrest and enter into mitosis. Taken together, these observations show that Pum and Nos are both required for the repression of the G2/M transition in the migrating pole cells (Asaoka-Taguchi, 1999).

Since pum and nos mutations do not affect posterior localization of maternal cyclin B mRNA or its partitioning into pole cells, it is concluded that translation of Cyclin B mRNA is usually repressed by Pum and Nos in pole cells. Cyclin B mRNA contains an NRE-like sequence in its 3' UTR, called the translation-control element (TCE). Deletion of the TCE from the 3' UTR of an epitope-tagged Cyclin B mRNA results in a phenotype similar to that caused by nos and pum mutations. These observations lead to the conclusion that Pum/Nos-dependent translational repression of cyclin B mRNA is mediated by the TCE. Given that Pum binds to the NRE in vitro, it is reasonable to suggest that Pum binds directly to the TCE. This is the first demonstration that maternal factors regulate the translation of specific mRNA in germline progenitors (Asaoka-Taguchi, 1999).

Repression of Pumilio protein expression by Rbfox1 promotes germ cell differentiation

RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The potential splicing-independent functions of these proteins remain unknown. This study demonstrates that cytoplasmic Drosophila Rbfox1, called such to conform to mammalian homologs, but given the FlyBase designation Ataxin-2 binding protein 1 (A2bp1), regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3'UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Misexpression of pumilio results in the formation of germline tumors, which contain cysts that break down and dedifferentiate back to single, mitotically active cells. Together, these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox-related diseases (Carreira-Rosario, 2016).

RNA-binding proteins play an integral role in mRNA metabolism, splicing, transport, and translation. An increasing number of studies link mutations in genes encoding RNA-binding proteins with a variety of diseases, highlighting the importance of these proteins with regard to human health. Rbfox proteins represent one such family and contain a highly conserved, centrally located RNA recognition motif (RRM) flanked by intrinsically disordered regions. Mammals have three Rbfox paralogs: RBFOX1 (A2BP1), RBFOX2 (RBM9) , and RBFOX3 (NeuN) . Nuclear isoforms of these genes regulate alternative splicing by directly binding to intronic (U)GCAUG elements, resulting in the exclusion or inclusion of downstream or upstream exons, respectively. In mice, disruption of Rbfox1 in neurons leads to neuronal hyperactivity, while loss of Rbfox2 results in cerebellum development defects. Rbfox1 and Rbfox2 have been implicated in a number of diseases including cancer, diabetes, and neurological disorders such as autism, mental retardation, and epilepsy. In all these examples, the observed phenotypes have been attributed to perturbations in normal mRNA splicing patterns (Carreira-Rosario, 2016).

Specific isoforms of Rbfox genes localize to the cytoplasm of cells in a variety of tissues across species. While the molecular functions of these isoforms remain poorly understood, both nuclear and cytoplasmic isoforms appear to act as tumor suppressors in the context of glioblastomas. Loss of cytoplasmic Rbfox1 has also been associated with colorectal cancer, and abnormal cytoplasmic inclusions of Rbfox1 are often observed in spinocerebellar ataxia type II patients. Recent studies have also shown that Rbfox proteins bind to many different 3'UTRs in the mammalian brain. These observations suggest that Rbfox proteins carry out additional functions beyond their established roles in splicing (Carreira-Rosario, 2016 and references therein).

The Drosophila genome contains a single Rbfox homolog called A2bp1, referred to as Rbfox1 to remain consistent with nomenclature across species. Mutations in the Drosophila Rbfox1 result in germline tumor formation. This study shows that cytoplasmic Rbfox1 is necessary for Drosophila germline development and regulates the stability and translation of specific mRNAs by binding to (U)GCAUG elements contained within their 3'UTR sequences. It was further shown that within the germline Rbfox1 targets pumilio to promote differentiation. Thus, this study reveals a splicing-independent function of Rbfox proteins, the disruption of which may contribute to RBFOX-linked diseases (Carreira-Rosario, 2016).

Rbfox family members from different species localize to either the nucleus or the cytoplasm. While isoforms that localize to the nucleus play a clear role in regulating alternative splicing, the function of cytoplasmic isoforms has remained less clear. Previous results showed that loss of Rbfox1 in Drosophila resulted in a block of germ cell differentiation. This study sought to determine the extent to which disruption of either nuclear or cytoplasmic Rbfox1 isoforms contributed to this phenotype. Transgenic rescue, isoform-specific RNAi knockdown and isoform-specific knockout experiments provide strong evidence that two cytoplasmic Rbfox1 isoforms specifically promote germ cell differentiation during the early stages of germline cyst development. The Drosophila genome does not encode for another redundant Rbfox family member. Thus the Drosophila ovary represents a unique platform on which to explore the function of cytoplasmic Rbfox family members in an in vivo setting (Carreira-Rosario, 2016).

Further experiments showed that cytoplasmic Rbfox1 regulates gene expression through a 3'UTR-dependent mechanism. The defining RRM domain of the Rbfox protein family is highly conserved across species. In vitro and in vivo experiments presented here indicate that Drosophila Rbfox1 physically associates with RNAs that contain GCAUG elements, similar to mammalian Rbfox proteins. Recent studies using RNA-crosslinking immunoprecipitation approaches have shown that mammalian Rbfox1, Rbfox2, and Rbfox3 all physically interact with 3'UTR sequences that contain GCAUG sites or other similar elements. The current experiments show that the presence of GCAUG sites within mRNA 3'UTRs results in modest decreases in mRNA stability, and more much dramatic decreases in protein expression. These observations suggest that Drosophila Rbfox1 acts to repress the translation of specific target mRNAs (Carreira-Rosario, 2016).

An increasing number of GCAUG sites within 3'UTRs appeared to have an additive effect on target gene expression in the context of germ cells. The presence of one site had little or no effect, at least in the context of the reporters that were used, while the presence of two or three sites resulted in a clear repression of protein expression in Rbfox1-expressing cells. While the repression of 3'UTR GCAUG reporters occurred in both the germline and within specific neurons, it remains possible that cytoplasmic Rbfox family members may regulate gene expression in a different manner in different contexts. For example, a newly published study shows that mammalian Rbfox proteins can promote the stability and translation of a target gene in cell culture. The functional significance of this regulation remains to be tested in vivo. Regardless, these findings, together with results presented here, indicate that the ability of cytoplasmic Rbfox family members to regulate protein expression has been conserved across species. The direction and degree of cytoplasmic Rbfox-dependent gene regulation may depend on different cell-specific proteins or on the presence of other 3'UTR regulatory elements within a given target transcript. The discovery of this function has significant implications for understanding of how Rbfox family members regulate normal development, as well as the disorders linked with disruption of Rbfox genes such as epilepsy, autism, and cancer (Carreira-Rosario, 2016).

The search for functionally relevant endogenous mRNA targets of Drosophila Rbfox1 led to the finding that Rbfox1 represses Pumilio protein expression during early germline cyst differentiation. Previous studies noted the presence of Pumilio protein in GSCs, cystoblasts, and two-cell cysts, but the mechanisms responsible for the stage-specific decrease of Pumilio expression in four-, eight-, and 16-cell cysts, and the functional significance of this expression pattern, have remained unknown. This study shows that Pumilio expression decreases as Rbfox1 expression increases. Examining the 3'UTR sequence of pumilio revealed the presence of four GCAUG sites, two of which showed extensive sequence conservation across many Drosophila species. Strikingly, Rbfox1, Rbfox2, and Rbfox3 also physically associate with Pumilio1 and Pumilio2 mRNA in the mouse nervous system (Carreira-Rosario, 2016).

Further analysis showed that Pumilio expression in the germline is regulated through a 3'UTR-dependent mechanism. A wild-type pumilio 3'UTR reporter exhibited an expression pattern similar to the endogenous protein, displaying decreased expression in the presence of Rbfox1. Mutating each of the four GCAUG elements within the pumilio 3'UTR sequence resulted in a striking expansion of reporter expression into the four-, eight-, and 16-cell cyst stages, suggesting that Rbfox1 negatively regulates Pumilio expression. qRT-PCR analysis of synchronously differentiating germ cells showed that endogenous pumilio mRNA levels increased in the absence of Rbfox1. These findings are in contrast to data obtained comparing the 3x Rbfox1 sensor to the mutant reporter, and suggest that Rbfox1 may influence the stability of specific target mRNAs in different contexts. Nonetheless, the degree to which Pumilio protein expression increases in the absence of Rbfox1 in these experiments is consistent with the model that Rbfox1 also regulates the expression of Pumilio, at least in part, at the level of translation. Other translational regulators, such as Bruno, also appear to influence mRNA stability (Carreira-Rosario, 2016).

The repression of Pumilio expression by Rbfox1 helps to promote germ cell differentiation. Loss of Rbfox1 results in germline tumor formation and an expansion of Pumilio expression. Strikingly, knockdown of pumilio strongly suppresses the Rbfox1 tumorous phenotype, leading to the formation of egg chambers with polyploid nuclei. While cytoplasmic Rbfox1 likely regulates the expression of other genes, the strength of this genetic interaction indicates that pumilio represents a major functional target of Rbfox1 in regard to germ cell differentiation. Mis-expression of a pumilio transgene, lacking the endogenous 3'UTR, in an otherwise wild-type background, phenocopies Rbfox1 mutants. These data indicate that germ cells must repress Pumilio expression before they can proceed into the next stage of development. Pumilio homologs are essential genes for germ cell maintenance across species. Given the conservation of Rbfox1-binding sites within the 3'UTR of pumilio mammalian homologs, repression of pumilio by Rbfox may represent a conserved mechanism that promotes germline differentiation (Carreira-Rosario, 2016).

Strikingly, morphological and molecular markers suggest that overexpression of Pumilio results in the dedifferentiation of germ cells. Rbfox1 mutants exhibit a similar phenotype. Pumilio overexpressing ovaries contain large tumors with multicellular cysts throughout their germaria. As these cysts continue to age and move toward the posterior of germaria, they begin to break down, as marked by the fragmentation of fusomes and ring canals. Similar observations have been made in both the Drosophila ovary and testis when germline cysts are experimentally prompted to undergo dedifferentiation. Furthermore, these germ cells re-acquire the expression of cytoplasmic Sxl, which typically marks GSCs, cystoblasts and two-cell cysts. Single cells derived from cyst breakdown remain mitotically active. These data indicate that germ cells must actively shut down gene expression programs that foster self-renewal and early differentiation before they can advance to the next stage of development. Failure to do so results in the reversion of the cells back to an earlier developmental state. It is anticipated that loss of Rbfox1, and the corresponding mis-expression of Rbfox1 target genes, may have similar effects in different tissues and in different species (Carreira-Rosario, 2016).


GENE STRUCTURE

cDNA clone length - 5952 and 6835 - There are two cDNAs, the 5' sequence being longer in one than in the other. They also differ in first exons and thus may have different promoters (Barker, 1992). Additionally, there are two different RNA species (Macdonald, 1992).

Bases in 5' UTR -883 and 1044

Exons - 12

Bases in 3' UTR - 2173


PROTEIN STRUCTURE

Amino Acids - 1533

Structural Domains

The pum gene is unusually large; comparison of genomic and cDNA sequences reveals that the pum transcription unit is at least 160 kb in length. The pum cDNA encodes a 157 x 10(3) M(r) protein which consists mainly of regions enriched in single amino acid repeats, usually glycine, alanine, glutamine or serine/threonine. Six tandem repeats of a 36 amino acid repeat unit are also present (Macdonald, 1992).

Pumilio is the prototypical member of an RNA-binding protein family evolutionarily conserved from yeast to humans. Its signature domain is termed a Puf motif after Drosophila Pumilio and the C. elegans translational regulator FBF (fem-3-binding factor). Puf proteins are implicated in post-transcriptional gene expression in S. cerevisiae, C. elegans, X. laevis and Drosophila. In most characterized situations, these proteins function with Nanos or Nanos-like partners (Gamberi, 2002 and references therein).


EVOLUTIONARY HOMOLOGS

Yeast Pumilio homologs

Eukaryotic post-transcriptional regulation is often specified by control elements within mRNA 3'- untranslated regions (3'-UTRs). In order to identify proteins that regulate specific mRNA decay rates in Saccharomyces cerevisae, the role of five members of the Puf family present in the yeast genome (referred to as JSN1/PUF1, PUF2, PUF3, PUF4 and MPT5/PUF5) was analyzed. Yeast strains lacking all five Puf proteins show differential expression of numerous yeast mRNAs. Examination of COX17 mRNA indicates that Puf3p specifically promotes decay of this mRNA by enhancing the rate of deadenylation and subsequent turnover. Puf3p also binds to the COX17 mRNA 3'-UTR in vitro. This indicates that the function of Puf proteins as specific regulators of mRNA deadenylation has been conserved throughout eukaryotes. In contrast to the case in Caenorhabditis elegans and Drosophila, yeast Puf3p does not affect translation of COX17 mRNA. These observations indicate that Puf proteins are likely to play a role in the control of transcript-specific rates of degradation in yeast by interacting directly with the mRNA turnover machinery (Olivas, 2000).

In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. To study the mechanism regulating ASH1 mRNA translation, the ASH1 locasome was isolated and the associated proteins were characterized by MALDI-TOF. One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression. Puf6p binds PUF consensus sequences in the 3'UTR of ASH1 mRNA and represses the translation of ASH1 mRNA both in vivo and in vitro. In the puf6Delta strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced. It is proposed that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed (Gu, 2004).

Drosophila Pumilio (Pum) and C. elegans FBF bind to the 3'-untranslated region (3'-UTR) of their target mRNAs and repress translation. Pum and FBF are members of a large and evolutionarily conserved protein family, the Puf family, found in Drosophila, C.elegans, humans and yeasts. Budding yeast, Saccharomyces cerevisiae, has five proteins with conserved Puf motifs: Mpt5/Uth4, Ygl014w, Yll013c, Jsn1 and Ypr042c. Mpt5 negatively regulates expression of the HO gene. Loss of MPT5 increases expression of reporter genes integrated into the ho locus, whereas overexpression of MPT5 decreases expression. Repression requires the 3'-UTR of HO, which contains a tetranucleotide, UUGU, also found in the binding sites of Pum and FBF. Mutation of UUGU to UACU in the HO 3'-UTR abolishes Mpt5-mediated repression. Studies using a three-hybrid assay for RNA binding indicate that Mpt5 binds to the 3'-UTR of HO mRNA containing a UUGU sequence but not a UACU sequence. These observations suggest that the yeast Puf homolog, Mpt5, negatively regulates HO expression post-transcriptionally (Tadauchi, 2001).

PUF proteins, a family of RNA-binding proteins, interact with the 3' untranslated regions (UTRs) of specific mRNAs to control their translation and stability. PUF protein action is commonly correlated with removal of the poly(A) tail of target mRNAs. This study focuses on how PUF proteins enhance deadenylation and mRNA decay. A yeast PUF protein physically binds Pop2p (Drosophila homolog Pop2), which is a component of the Ccr4p-Pop2p-Not deadenylase complex, and Pop2p is required for PUF repression activity. By binding Pop2p, the PUF protein simultaneously recruits the Ccr4p deadenylase (homolog of Drosophila Twin) and two other enzymes involved in mRNA regulation, Dcp1p and Dhh1p. Regulated deadenylation was reconstituted in vitro and it was demonstrated that the PUF-Pop2p interaction is conserved in yeast, worms and humans. It is suggested that the PUF-Pop2p interaction underlies regulated deadenylation, mRNA decay and repression by PUF proteins (Goldstrohm, 2006).

Invertebrate Pumilio homologs

The nematode Caenorhabditis elegans has two sexes: males and hermaphrodites. Hermaphrodites Initially produce sperm but switch to producing oocytes. This switch appears to be controlled by the 3' untranslated region of fem-3 messenger RNA. A binding factor (FBF) has been identified that is a cytoplasmic protein that binds specifically to the regulatory region of fem-3 3'UTR and mediates the sperm/oocyte switch. The RNA-binding domain of FBF consists of a stretch of eight tandem repeats and two short flanking regions. This structural element is conserved in several proteins including Drosophila Pumilio, a regulatory protein that controls pattern formation in the fly by binding to a 3'UTR. It is proposed that FBF and Pumilio are members of a widespread family of sequence-specific RNA-binding proteins (Zhang, 1997).

The Caenorhabditis elegans FBF protein and its Drosophila relative, Pumilio, define a large family of eukaryotic RNA-binding proteins. By binding regulatory elements in the 3' untranslated regions (UTRs) of their cognate RNAs, FBF and Pumilio have key post-transcriptional roles in early developmental decisions. In C. elegans, FBF is required for repression of fem-3 mRNA to achieve the hermaphrodite switch from spermatogenesis to oogenesis. FBF and NANOS-3 (NOS-3), one of three C. elegans Nanos homologs, interact with each other in both yeast two-hybrid and in vitro assays. The portions of each protein required for this interaction have been delineated. Worms lacking nanos function were derived either by RNA-mediated interference (nos-1 and nos-2) or by use of a deletion mutant (nos-3). The roles of the three nos genes overlap during germ-line development. In certain nos-deficient animals, the hermaphrodite sperm-oocyte switch is defective, leading to the production of excess sperm and no oocytes. In other nos-deficient animals, the entire germ line dies during larval development. This germ-line death does not require CED-3, a protease required for apoptosis. The data suggest that NOS-3 participates in the sperm-oocyte switch through its physical interaction with FBF, forming a regulatory complex that controls fem-3 mRNA. NOS-1 and NOS-2 also function in the switch, but do not interact directly with FBF. The three C. elegans nanos genes, like Drosophila nanos, are also critical for germ-line survival. It is proposed that this may have been the primitive function of nanos genes (Kraemer, 1999).

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF ('fem-3 mRNA binding factor'), were originally discovered as regulators of germline sex determination. FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. It is suggested that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family ('Pumilio and FBF') of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. It is suggested that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells (Crittenden, 2002).

In the C. elegans germline, GLP-1/Notch signaling and two nearly identical PUF (Pumilio and FBF) protein family RNA binding proteins, FBF-1 and FBF-2, promote proliferation. Here, the fbf-1 and fbf-2 genes are largely redundant for promoting mitosis but they have opposite roles in fine-tuning the size of the mitotic region. The mitotic region is smaller than normal in fbf-1 mutants but larger than normal in fbf-2 mutants. Consistent with gene-specific roles, fbf-2 expression is limited to the distal germline, while fbf-1 expression is broader. The fbf-2 gene, but apparently not fbf-1, is controlled by GLP-1/Notch signaling, and the abundance of FBF-1 and FBF-2 proteins is limited by reciprocal 3′UTR repression. It is proposed that the divergent fbf genes and their regulatory subnetwork enable a precise control over size of the mitotic region. Therefore, fbf-1 and fbf-2 provide a paradigm for how recently duplicated genes can diverge to fine-tune patterning during animal development (Lamont, 2004).

RNA binding proteins are key regulators of the germline decision between proliferation and differentiation. Of particular importance to this paper are FBF-1 and FBF-2 (for fem-3 Binding Factor) -- two nearly identical regulators of the PUF family. The FBF-1 and FBF-2 proteins are collectively called FBF, and similarly, fbf-1 and fbf-2 are collectively called the fbf genes. The nucleotide sequences of fbf-1 and fbf-2 are 93% identical, and the amino acid sequences are 91% identical, suggesting that fbf-1 and fbf-2 are recently duplicated genes. During early larval stages, germline proliferation is normal in fbf-1 fbf-2 double mutants, but in the fourth larval stage, the germline precociously leaves the mitotic cell cycle to enter meiosis and differentiate as sperm. In addition, depletion of both fbf-1 and fbf-2 eliminates the hermaphrodite switch from spermatogenesis to oogenesis. Therefore, FBF is required for continued mitotic divisions in the germline as well as for the hermaphrodite sperm/oocyte switch (Lamont, 2004).

PUF proteins bind specifically to regulatory elements, usually in the 3' untranslated region (UTR) of a target mRNA, and repress that mRNA, either by promoting mRNA degradation or inhibiting translation. Pumilio, for example, inhibits translation of hunchback mRNA in the early Drosophila embryo, whereas PUF-5/Mpt5 destabilizes HO mRNA in yeast. In C. elegans, FBF-1 and FBF-2 promote mitosis by repressing mRNAs that encode regulators critical for entry into the meiotic cell cycle, and they promote the sperm/oocyte switch by repressing the fem-3 sperm-promoting mRNA. Both FBF-1 and FBF-2 bind specifically to the same RNA target sequence, which differs from the Pumilio binding site. The molecular mechanism by which FBF represses mRNAs in the C. elegans germline remains unknown, but by analogy with its homologs in yeast and Drosophila, FBF is likely to control the stability or translation of its target mRNAs (Lamont, 2004).

Previous studies have suggested that FBF-1 and FBF-2 are redundant: fbf-1 single mutants are grossly normal, albeit with smaller mitotic regions and more hermaphrodite sperm than wild-type. This study confirms the fbf-1/fbf-2 redundancy but also identify individual roles for each gene in regulating the size of the mitotic region. Like fbf-1, the fbf-2 single mutants are grossly normal, but in contrast to fbf-1, fbf-2 mutant germlines have a larger mitotic region than normal and can be feminized. Consistent with fbf-1 and fbf-2 having individual roles, their mRNAs and proteins are expressed in distinct patterns. Furthermore, the fbf-2 gene appears to be a direct target of GLP-1/Notch signaling, a finding that forges the first molecular link between GLP-1/Notch signaling and the RNA regulatory circuit. fbf-1 and fbf-2 repress each other's expression and this reciprocal repression is likely to be direct via FBF binding sites in the fbf-1 and fbf-2 3' UTRs. It is suggested that GLP-1/Notch signaling and FBF autoregulation work together to control the distribution and amount of FBF and thereby fine-tune the size of the mitotic region (Lamont, 2004).

FBF-1 and FBF-2 (collectively FBF) are two nearly identical Puf-domain RNA-binding proteins that regulate the switch from mitosis to meiosis in the C. elegans germline. In germline stem cells, FBF prevents premature meiotic entry by inhibiting the expression of meiotic regulators, such as the RNA-binding protein GLD-1. This study demonstrates that FBF also directly inhibits the expression of structural components of meiotic chromosomes. HIM-3, HTP-1, HTP-2, SYP-2 and SYP-3 are components of the synaptonemal complex (SC) that forms between homologous chromosomes during meiotic prophase. In wild-type germlines, the five SC proteins are expressed shortly before meiotic entry. This pattern depends on FBF binding sites in the 3' UTRs of the SC mRNAs. In the absence of FBF or the FBF binding sites, SC proteins are expressed precociously in germline stem cells and their precursors. SC proteins aggregate and SC formation fails at meiotic entry. Precocious SC protein expression is observed even when meiotic entry is delayed in fbf mutants by reducing GLD-1. It is proposed that parallel regulation by FBF ensures that in wild-type gonads, meiotic entry is coordinated with just-in-time synthesis of synaptonemal proteins (Merritt, 2010).

Since stem cells are rare and difficult to study in vivo in adults, the use of classical models of regeneration to address fundamental aspects of the stem cell biology is emerging. Planarian regeneration, which is based upon totipotent stem cells present in the adult – the so-called neoblasts – provides a unique opportunity to study in vivo the molecular program that defines a stem cell. The choice of a stem cell to self-renew or differentiate involves regulatory molecules that also operate as translational repressors, such as members of PUF proteins. In this study, a homologue of the Drosophila PUF gene Pumilio (DjPum) was identified in the planarian Dugesia japonica, with an expression pattern preferentially restricted to neoblasts. Through RNA interference (RNAi), gene silencing of DjPum was demonstrated to dramatically reduces the number of neoblasts, thus supporting the intriguing hypothesis that stem cell maintenance may be an ancestral function of PUF proteins (Salvetti, 2005).

A conserved PUF-Ago-eEF1A complex attenuates translation elongation

PUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar, yet distinct, mechanisms. This study reports that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode (C. elegans) and mammalian PUF-Ago-eEF1A complexes were identified, using coimmunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promoted repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1-CSR-1 heterodimer inhibited EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2-Ago-eEF1A inhibited translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurred after translation initiation and led to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerged from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF-Ago-eEF1A complex attenuates translation elongation (Friend, 2012).

Xenopus Pumilio homologs

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, it has been shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes. This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments have shown in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB: Drosophila homolog Orb), a key regulator of translational repression and activation of mRNAs stored in oocytes. This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates (Nakahata, 2001).

Drosophila Pumilio and a C. elegans Pumilio homolog, FBF, are members of the Pumilio-homology domain (Pum-HD) family, also known as thePuf (for Pumilio and FBF) family. The sequence in the C-terminal region of the Pum-HD family is highly conserved in many species, including human homologs (DDBJ/EMBL/GenBankTM accession numbers KIAA0099 and KIAA0235) deduced from their cDNAs. A Xenopus 2.0-kb sequence (DDBJ/EMBL/GenBankTM accession number AB045628) was obtained that contained a domain equivalent to those of Drosophila Pumilio (78% identity) and the human homolog KIAA0099 (95% identity). This domain is known as the diagnostic hallmark of the Pum-HD family and is defined by the presence of eight copies of an imperfect repeat sequence, comprising a specific RNA-binding domain (Nakahata, 2001).

The actual biological roles of XPum are completely unknown at present, but it can be speculated that XPum plays an important role in translational control of cyclin B1 mRNA, as in Drosophila. CPEB directly binds to maskin, a protein that can also bind directly to the cap-binding translation initiation factor elF-4E, which leads to translational repression. The dissociation of maskin from elF-4E allows elF-4G to bind to elF-4E, which brings elF-3 and the 40 S ribosomal subunit to the mRNA to initiate translation via cap-ribose methylation. Recent studies have also shown that a progesterone-induced early phosphorylation of CPEB at serine 174 is catalyzed by Eg2 and that this phosphorylation recruits cleavage and polyadenylation specificity factor into an active cytoplasmic polyadenylation complex. Thus, CPEB plays a key role in both translational repression and activation of mRNAs stored in oocytes. XPum is physically associated with CPEB in oocytes. In cooperation with CPEB, XPum may control the CPEB/maskin-mediated translational masking and unmasking to assure the highly coordinated successive translational activation of masked mRNAs during oocyte maturation. Further studies are required to understand the biological significance of the interactions among XPum, CPEB, and cyclin B1 mRNA, as well as to elucidate the functions of XPum in oocytes (Nakahata, 2001).

Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In Xenopus it has been shown that the cytoplasmic polyadenylation element (CPE) in the 3'-untranslated region (UTR) of cyclin B1 mRNA is responsible for both translational repression (masking) and activation (unmasking) of the mRNA (Mendez and Richter, 2001; Richter, 2000). The CPE is bound by a CPE-binding protein. In this study, the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, was investigated in mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound, in mature oocytes, to the unphosphorylated form of cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. It is proposed that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation (Nakahata, 2003).

One possible mechanism of translational activation of cyclin B1 mRNA is that a dissociation of XPum from phosphorylated CPEB during oocyte maturation induces destabilization of the CPEB-maskin-eIF4E complex and provides a cue that leads to unmasking of cyclin B1 mRNA by the mechanism common to CPE-containing mRNAs. In this respect, it is noteworthy that phosphorylation of CPEB on Ser210, which occurs about the time of cyclin B1 translation, is sufficient for selective translational activation of cyclin B1. While this phenomenon has been explained in relation to degradation of CPEB, it is also conceivable that the later phosphorylation of CPEB induces release of XPum from the CPEB-maskin-eIF4E complex and that this event triggers translational activation of cyclin B1. Consistent with this possibility, it has been demonstrated that phosphorylation of CPEB is required for its dissociation from a large ribonucleoprotein complex upon oocyte maturation, prior to degradation (Nakahata, 2003).

Mammalian Pumilio homologs

Puf proteins are developmental regulators that control mRNA stability and translation by binding sequences in the 3' untranslated regions of their target mRNAs. The structure of the RNA binding domain of the human Puf protein, Pumilio1, has been determined, bound to a high-affinity RNA ligand. The RNA binds the concave surface of the molecule, where each of the protein's eight repeats makes contacts with a different RNA base via three amino acid side chains at conserved positions. These three side chains were mutated in one repeat, thereby altering the sequence specificity of Pumilio1. Thus, the high affinity and specificity of the PUM-HD for RNA is achieved using multiple copies of a simple repeated motif (Wang, 2002).

Germ cell development is complex; it encompasses specification of germ cell fate, mitotic replication of early germ cell populations, and meiotic and postmeiotic development. Meiosis alone may require several hundred genes, including homologs of the BOULE (BOL) and PUMILIO (PUM) gene families. Both BOL and PUM homologs encode germ cell specific RNA binding proteins in diverse organisms where they are required for germ cell development. Human BOL forms homodimers and is able to interact with a PUMILIO homolog, PUM2. The domain of BOL that is required for dimerization and for interaction with PUM2 was mapped. BOL and PUM2 can form a complex on a subset of PUM2 RNA targets that is distinct from targets bound by PUM2 and another deleted in azoospermia (DAZ) family member, DAZ-like (DAZL). This suggests that RNA sequences bound by PUM2 may be determined by protein interactions. This data also suggests that although the BOL, DAZ, and DAZL proteins are all members of the same gene family, they may function in distinct molecular complexes during human germ cell development (Urano, 2005).

Members of the Pumilio and DAZL family of RNA binding proteins are required for germ cell development in Drosophila, Xenopus, and Caenorhabditis elegans. This study reports identification and characterization of RNA sequences to which PUM2 and DAZL bind. Human PUM2 specifically recognizes the Drosophila Pumilio RNA target (the NRE or Nanos regulator element sequence); single nucleotide changes in the NRE abolished PUM2 binding. Then, coimmunoprecipitation was used to isolate human transcripts specifically bound by PUM2 and DAZL and subsequently those were identified that contain NRE-like sequence elements. The interacting proteins, PUM2 and DAZL, are capable of binding the same RNA target and mRNA sequences bound by both proteins in the 3'UTR of human SDAD1 mRNA were further characterized. Taken together, the results define sequences to which these germ cell-specific RNA binding proteins may bind to promote germ cell development (Fox, 2005).

Pumilio (Pum) protein acts as a translational inhibitor in several organisms including yeast, Drosophila, Xenopus, and mammals. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals, but their function in neurons has not been identified. In this study, it was found that Pum2 mRNA is expressed during neuronal development and that the protein is found in discrete particles in both the cell body and the dendritic compartment of fully polarized neurons. This finding indicates that Pum2 is a novel candidate of dendritically localized ribonucleoparticles (RNPs). During metabolic stress, Pum2 is present in stress granules (SGs), which are subsequently detected in the somatodendritic domain. It remains excluded from processing bodies under all conditions. When overexpressed in neurons and fibroblasts, Pum2 induces the formation of SGs that also contain T-cell intracellular antigen 1 (TIA-1)-related protein, eukaryotic initiation factor 4E, poly(A)-binding protein, TIA-1, and other RNA-binding proteins including Staufen1 and Barentsz. This induction of SGs is dependent on the RNA-binding domain and a glutamine-rich region in the N terminus of Pum2. This glutamine-rich region behaves in a similar manner as TIA-1 and prion protein, two molecules with known roles in protein aggregation. Pum2 downregulation in neurons via RNA interference (RNAi) interferes with the formation of SGs during metabolic stress. Cotransfection with an RNAi-resistant portion of the Pum2 mRNA restores SG formation. These results suggest a role for Pum2 in dendritic RNPs and SG formation in mammalian neurons (Vessey, 2006).

Key regulators of 3' untranslated regions (3' UTRs) are microRNAs and RNA-binding proteins (RBPs). The p27 tumour suppressor is highly expressed in quiescent cells, and its downregulation is required for cell cycle entry after growth factor stimulation. Intriguingly, p27 accumulates in quiescent cells despite high levels of its inhibitors miR-221 and miR-222. This study shows that miR-221 and miR-222 are underactive towards p27-3' UTR in quiescent cells, as a result of target site hindrance. Pumilio-1 (PUM1) is a ubiquitously expressed RBP that was shown to interact with p27-3' UTR. In response to growth factor stimulation, PUM1 is upregulated and phosphorylated for optimal induction of its RNA-binding activity towards the p27-3' UTR. PUM1 binding induces a local change in RNA structure that favours association with miR-221 and miR-222, efficient suppression of p27 expression, and rapid entry to the cell cycle. This study has therefore uncovered a novel RBP-induced structural switch modulating microRNA-mediated gene expression regulation (Kedde, 2010).

Temporal control of messenger RNA (mRNA) translation is an important mechanism for regulating cellular, neuronal, and developmental processes. However, mechanisms that coordinate timing of translational activation remain largely unresolved. Full-grown oocytes arrest meiosis at prophase I and deposit dormant mRNAs. Of these, translational control of cyclin B1 mRNA in response to maturation-inducing hormone is important for normal progression of oocyte maturation, through which oocytes acquire fertility. This study found that dormant cyclin B1 mRNA forms granules in the cytoplasm of zebrafish and mouse oocytes. Real-time imaging of translation revealed that the granules disassemble at the time of translational activation during maturation. Formation of cyclin B1 RNA granules requires binding of the mRNA to Pumilio1 protein and depends on actin filaments. Disruption of cyclin B1 RNA granules accelerates the timing of their translational activation after induction of maturation, whereas stabilization hinders translational activation. Thus, these results suggest that RNA granule formation is critical for the regulation of timing of translational activation (Kotani, 2013).


pumilio: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 August 2011

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