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C. elegans Bruno homolog
While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using C. elegans as a model for studying neuron/target communication, this study reports that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. Human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, a new neuronal, putative pre-mRNA splicing factor, UNC-75, has been identified and it has been shown that UNC-75, as well as the C. elegans homolog of ELAV, are each required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function (Loria, 2003).
The vertebrate orthologs of UNC-75, CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5, have been shown to be involved in splicing in an in vitro assay. To investigate whether the function of these proteins is conserved (a notion that was expected from the level of primary sequence similarity), UNC-75 and its human orthologs were compared in more detail. mRNA samples derived from a variety of different human tissues were hybridized with probes specific to three of the four human orthologs. CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5 each show highly similar expression patterns that are largely restricted to the nervous system. Within the nervous system, every region tested shows expression of CELF3/BrunoL1, CELF4/BrunoL4, and CELF5/BrunoL5. Thus, the pan-neuronal expression of the human orthologs of UNC-75 mirrors the pan-neuronal expression of C. elegans UNC-75 (Loria, 2003).
In addition to their similar tissue distribution, the function and subcellular localization of the worm and human proteins are also conserved. A human CELF4/BrunoL4 cDNA, expressed under control of the unc-75 promoter, is able to rescue the uncoordinated phenotype of unc-75 mutants as well as the resistance to the acetylcholine esterase inhibitor aldicarb ('ric' phenotype), which likely results from a disruption in signaling between cholinergic ventral cord motor neurons and their body wall muscle targets. Furthermore, translational fusions of CELF4/BrunoL4 with GFP (punc75::gfp::L4) show localization to subnuclear speckles reminiscent of the UNC-75::GFP speckles. Since loss of UNC-75 can be rescued by a human ortholog that acts as a splicing factor in vitro, it is reasonable to suggest that UNC-75 also acts as a pre-mRNA splicing factor, a notion further corroborated by its subnuclear localization (Loria, 2003).
Functional comparison of UNC-75 and EXC-7 proteins was extended by analyzing exc-7 null mutant animals. In contrast to fly Elav, which severely affects neuronal development and viability, exc-7 mutants are viable and show no locomotory or defecation defects. Also in contrast to fly Elav, exc-7 is only expressed in a subset of neurons in the nervous system, several of which are cholinergic neurons. The development and morphology of several cholinergic neuron classes were assessed by using cell-specific gfp markers; no obvious defects were found. Moreover, synaptic vesicles in the cholinergic SAB neurons cluster normally in exc-7 null mutants, leading to the conclusion that EXC-7 has no significant impact on neuronal development. However, when cholinergic motorneuron function was tested in more detail, it was found that exc-7(rh252) animals show a synaptic transmission defect similar to unc-75 (Loria, 2003).
The relation of the ric phenotype of unc-75 and exc-7 was assessed. If these two genes act in a similar process, their null phenotypes should not enhance one another. It was found, however, that the synaptic transmission defect of the double mutant is significantly enhanced compared to the single mutants. Moreover, although exc-7 mutant animals show no locomotory defects on their own, unc-75; exc-7 double mutant animals are smaller and appear significantly more uncoordinated than unc-75 single mutants. Lastly, it was found that the ric phenotype of exc-7 null mutants is not rescued by an elevation in ambient temperature. This lack of temperature sensitivity is similar to that of unc-17 mutants, which are affected in synaptic vesicle loading. It is concluded that unc-75 and exc-7 have nonredundant and distinct roles in cholinergic synaptic transmission and likely regulate the pre-mRNA splicing of a distinct set of target genes (Loria, 2003).
The regenerative abilities of freshwater planarians are based on neoblasts, stem cells maintained throughout the animal's life. A member of the Bruno-like family of RNA binding proteins is critical for regulating neoblasts in the planarian Schmidtea mediterranea. Smed-bruno-like (bruli) mRNA and protein are expressed in neoblasts and the central nervous system. Following bruli RNAi, which eliminates detectable bruli protein, planarians initiate the proliferative response to amputation and form small blastemas but then undergo tissue regression and lysis. The neoblast population was characterized by using antibodies recognizing SMEDWI-1 and Histone H4 (monomethyl-K20) and cell-cycle markers to label subsets of neoblasts and their progeny. bruli knockdown results in a dramatic reduction/elimination of neoblasts. These analyses indicate that neoblasts lacking bruli can respond to wound stimuli and generate progeny that can form blastemas and differentiate; yet, they are unable to self-renew. These results suggest that bruli is required for stem cell maintenance (Guo, 2006).
Bruno-like is involved in asymmetric distribution of maternal mRNAS in zebrafish
Asymmetric distribution of maternal mRNAs has not been well documented in
zebrafish. dazl mRNA is localized at the vegetal
pole. A novel zebrafish gene, bruno-like (brul) is described that provides another example of vegetal mRNA localization. brul encodes an Elav-type
RNA-binding protein that belongs to the Bruno-like family that includes
mammalian CUG-BP, Xenopus EDEN-BP, and Drosophila Bruno. At 24 hpf, brul mRNA
is abundant in lens fiber cells. At the onset of embryogenesis, maternal brul
mRNA is detected at the vegetal pole, and it then migrates rapidly toward the
blastoderm through yolk cytoplasmic streams. During oogenesis, brul mRNA becomes
localized at the vegetal cortex at stage II, later than dazl mRNA. Anchoring of brul mRNA was dependent on microfilaments (Suzuki, 2000).
etr-1, a possible Bruno homolog in Xenopus
In Xenopus development, dorsal mesoderm is thought to play a key role in both the induction and patterning of the nervous system. Noggin, which is expressed in dorsal mesoderm, can mimic that tissue's neural-inducing activity without inducing mesoderm. Neural tissue induced in ectodermal explants by noggin has been further characterized using four neural-specific genes: two putative RNA-binding proteins, nrp-1 and etr-1; the synaptobrevin sybII; and the lipocalin cpl-1. The expression domain of each gene during embryogenesis was determined and then expression of these genes was examined in noggin-treated explants. All markers, including the differentiated marker sybII, were expressed in noggin-induced neural tissue. cpl-1, a marker of dorsal brain, and etr-1, a marker absent in much of the dorsal forebrain, were both expressed in non-overlapping territories within these explants. It is concluded that the despite the absence of
mesoderm, noggin-induced neural tissue shows considerable differentiation and organization, which
may represent dorsal-ventral patterning of the forebrain (Knecht, 1995).
Mammalian Bruno homologs
Expansion of trinucleotides repeats is associated with a number of neurodegenerative diseases. Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease associated with a
(CTG)n nucleotide repeat expansion in the 3'-untranslated region of the myotonin protein kinase (Mt-PK) gene. These CTG repeats are translated into CUG repeats in messenger RNA. While an unstable CTG triplet repeat expansion is responsible for myotonic dystrophy, the mechanism
by which this genetic defect induces the disease remains unknown. To detect proteins binding to CTG
triplet repeats, bandshift analysis was performed using as probes double-stranded DNA fragments
having CTG repeats [ds(CTG)6-10] and single-stranded oligonucleotides having CTG repeats
ss(CTG)8 or RNA CUG triplet repeats (CUG)8. The source of protein was nuclear and cytoplasmic
extracts of HeLa cells, fibroblasts and myotubes. Proteins binding to the double-stranded DNA repeat
[ds(CTG)6-10] are inhibited by nonlabeled ds(CTG)6-10, but not by a non-specific DNA fragment
(USF/AD-ML). Another protein binding to ssCTG probe and RNA CUG probe is inhibited by
nonlabeled (CTG)8 and (CUG)8. Nonlabeled oligos with different triplet repeat sequences, either ss(CAG)8
or ss(CGG)8, do not inhibit binding to the ss(CTG)8 probe. However, when labeled as probes, the
(CAG)8 and (CGG)8 bind to proteins distinct from the CTG proteins and binding is inhibited by
nonlabeled (CAG)8 or (CGG)8 respectively. The protein binding only to the RNA repeat (CUG)8 is
inhibited by nonlabeled (CUG)8 but not by nonlabeled single- or double-stranded CTG repeats.
The CUG-BP exhibits no binding to an RNA oligonucleotide of triplet repeats of the
same length but having a different sequence, CGG. The CUG binding protein is localized to the
cytoplasm, whereas dsDNA binding proteins are localized to the nuclear extract. Thus, several
trinucleotide binding proteins exist and their specificity is determined by the triplet sequence. The novel
protein, CUG-BP, is particularly interesting since it binds to triplet repeats known to be present in
myotonin protein kinase mRNA, which is responsible for myotonic dystrophy (Timchenko, 1996a).
This study reports the isolation and characterization of a (CUG)n triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based on their ability to bind specifically to (CUG)8 oligonucleotides in vitro. While CUG-BP1 is the major (CUG)8-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is associated with polyadenylated RNAs in vivo. In vitro RNA-binding/photocrosslinking studies demonstrate that
CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3'-UTR. It is proposed that the (CUG)n repeat
region in Mt-PK mRNA is a binding site for CUG-BP/hNab50 in vivo, and triplet repeat expansion
leads to sequestration of this hnRNP on mutant Mt-PK transcripts (Timchenko, 1996b).
Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of
the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the
(CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. A protein has been isolated with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. The phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patients. CUG-BP/hNab50 is a substrate for DMPK (Drosophila homolog: Genghis Khan) both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK ( homozygous DM patients and DMPK knockout mice) shows that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in both the DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patients. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of
CUG-BP/hNab50 results in the dramatic reduction of CUG-BP2, the hypophosphorylated isoform,
accumulation of which is observed in the nuclei of DMPK knockout mice. These data suggest a
feedback mechanism whereby decreased levels of DMPK could alter the phosphorylation status of
CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. These results suggest that
DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of
lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs (Roberts, 1997).
The post-transcriptional regulation of gene expression by RNA-binding proteins is an important element in controlling both normal cell functions and animal development. The diverse roles are demonstrated by the Elav family of RNA-binding proteins, where various members have been shown to regulate several processes involving mRNA. Another family of RNA-binding proteins distantly related to the Elav family but closely related to Bruno, a translational regulator in Drosophila melanogaster, has been discovered. In humans, six Bruno-like genes have been identified, whereas other species such as Drosophila, Xenopus laevis, and Caenorhabditis elegans have at least two members of this family, and related genes have also been detected in plants and ascidians (phylum Urochordata). The human BRUNOL2 and BRUNOL3 are 92% identical in the RNA-binding domains, although the BRUNOL2 gene is expressed ubiquitously whereas BRUNOL3 is expressed predominantly in the heart, muscle, and nervous system. Both of these proteins bind the same target RNA, the Bruno response element. The RNA-binding domain that recognizes the Bruno response element is composed of two consecutive RNA recognition motifs at the amino terminus of vertebrate Bruno protein. The possible involvement of the Bruno family of proteins in the CUG repeat expansion disease, myotonic dystrophy, is discussed (Good, 2000).
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