InteractiveFly: GeneBrief

lin-28: Biological Overview | References


Gene name - lin-28

Synonyms -

Cytological map position - 64E1-64E1

Function - RNA-binding protein

Keywords - regulation of microRNA maturation, regulation of developmental timing, oogenesis, muscle development

Symbol - lin-28

FlyBase ID: FBgn0035626

Genetic map position - chr3L:5,647,783-5,650,484

Classification - Cold-Shock Protein - Ribosomal protein S1-like RNA-binding domain

Cellular location - possibly cytoplasmic and nuclear



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Narbonne-Reveau, K., Lanet, E., Dillard, C., Foppolo, S., Chen, C. H., Parrinello, H., Rialle, S., Sokol, N. S. and Maurange, C. (2016). Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila. Elife 5 [Epub ahead of print]. PubMed ID: 27296804
Summary:
Pediatric neural tumors are often initiated during early development and can undergo very rapid transformation. However, the molecular basis of this early malignant susceptibility remains unknown. During Drosophila development, neural stem cells (NSCs) divide asymmetrically and generate intermediate progenitors that rapidly differentiate in neurons. Upon gene inactivation, these progeny can dedifferentiate and generate malignant tumors. This study found that intermediate progenitors, are prone to malignancy only when born during an early window of development, during early larval stages, while expressing the transcription factor Chinmo, and the mRNA-binding proteins Imp/IGF2BP and Lin-28. These genes compose an oncogenic module that is coopted upon dedifferentiation of early-born intermediate progenitors to drive unlimited tumor growth. In late larvae, temporal transcription factor progression in NSCs silences the module, thereby limiting mitotic potential and terminating the window of malignant susceptibility. Thus, this study identifies the gene regulatory network that confers malignant potential to neural tumors with early developmental origins.
Luhur, A., Buddika, K., Ariyapala, I. S., Chen, S. and Sokol, N. S. (2017). Opposing post-transcriptional control of InR by FMRP and LIN-28 adjusts stem cell-based tissue growth. Cell Rep 21(10): 2671-2677. PubMed ID: 29212015
Summary:
Although the intrinsic mechanisms that control whether stem cells divide symmetrically or asymmetrically underlie tissue growth and homeostasis, they remain poorly defined. This study reports that the RNA-binding protein fragile X mental retardation protein (FMRP) limits the symmetric division, and resulting expansion, of the stem cell population during adaptive intestinal growth in Drosophila. The elevated insulin sensitivity that FMRP-deficient progenitor cells display contributes to their accelerated expansion, which is suppressed by the depletion of insulin-signaling components. This FMRP activity is mediated solely via a second conserved RNA-binding protein, LIN-28, known to boost insulin signaling in stem cells. Via LIN-28, FMRP controls progenitor cell behavior by post-transcriptionally repressing the level of Insulin receptor (InR). This study identifies the stem cell-based mechanism by which FMRP controls tissue adaptation, and it raises the possibility that defective adaptive growth underlies the accelerated growth, gastrointestinal, and other symptoms that affect fragile X syndrome patients.

BIOLOGICAL OVERVIEW

Understanding the control of stem cell (SC) differentiation is important to comprehend developmental processes as well as to develop clinical applications. Lin28 is a conserved molecule that is involved in SC maintenance and differentiation by regulating let-7 miRNA maturation. However, little is known about the in vivo function of Lin28. This study reports critical roles for lin-28 during oogenesis. let-7 maturation was shown to be increased in lin-28 null mutant fly ovaries. lin-28 null mutant female flies display reduced fecundity, due to defects in egg chamber formation. More specifically, in mutant ovaries, the egg chambers were shown to fuse during early oogenesis resulting in abnormal late egg chambers. This phenotype is the combined result of impaired germline SC differentiation and follicle SC differentiation. A model is suggested in which these multiple oogenesis defects result from a misregulation of the ecdysone signaling network, through the fine-tuning of Abrupt and Fasciclin2 expression. These results give a better understanding of the evolutionarily conserved role of lin-28 on GSC maintenance and differentiation (Stratoulias, 2014).

The Cold-Shock Domain (CSD) protein Lin28 was initially identified in Caenorhabditis elegans (C. elegans) as a component of the heterochronic pathway that regulates the timing of cell fate specification (Ambros, 1984). Subsequent discovery of gene expression regulation through small non-coding RNAs clarified the role of Lin28 in this pathway (Fire, 1998). The lin-28 mRNA is a conserved target of the let-7 micro-RNA (miRNA) family both in C. elegans and vertebrates. On the other hand, Lin28 inhibits let-7 processing (Viswanathan, 2008). At the molecular level, Lin28 protein interacts with the let-7 precursor (pre-let-7), resulting in inhibition of let-7 maturation (Piskounova, 2011). The let-7 inhibition occurs through the physical interaction of the pre-let-7 loop and Lin28 protein, preventing further processing of pre-let-7 towards the mature form of let-7 (Newman, 2008; Loughlin, 2012). Together, these interactions create a feedback loop between Lin28 and let-7, leading to a strict regulation of let-7 maturation (Zhong; 2010; Stratoulias, 2014 and references therein).

Lin28 raised further interest when it was used, along with Nanog, to replace the factors c-Myc and Klf4 in somatic cell reprogramming (Yu, 2007). These experiments, together with data from human embryonic stem cells, underscored the important role of lin-28 in pluripotency regulation and maintenance. Besides acting as a negative regulator of let-7 maturation, Lin28 has also been shown to have a direct effect on translation through the recruitment of the RNA Helicase A (Jin, 2011). This mode of function, independent of let-7 maturation, has been demonstrated in the case of Insulin-like Growth Factor 2 during mouse myogenesis. Lin28 binding on IGF-2 mRNA increases its translation efficiency and therefore facilitates skeletal myogenesis in mice (Polesskaya, 2007; Stratoulias, 2014 and references therein).

The Lin28 protein is composed of four domains: a positively charged linker that binds two Cys-Cys-His-Cys (CCHC)-type zinc-binding motifs to the CSD. In mammalian genomes, two paralogs of lin-28 are found, Lin28A and Lin28B. While Lin28B represses let-7 processing in the nucleus to prevent the formation of the precursor form from the primary let-7, Lin28A also blocks cytoplasmic processing of let-7 (Piskounova, 2011). It has recently been shown in mouse that deletion of the Lin28 linker domain alters the protein’s three-dimensional structure and is sufficient to disrupt sequestration of the precursor form of let-7 (pre-let-7) (Stratoulias, 2014).

The miRNA let-7 family is conserved across diverse animals, functioning to control late temporal transitions during development. During the last decade, the involvement of let-7 in regulating cell differentiation has been analyzed in various contexts, including neural cell specification, stem cell maintenance and hematopoietic progenitor differentiation. While eight different let-7 miRNA genes are annotated in the human genome, only one is found in Drosophila melanogaster. Like in C. elegans, in Drosophila the loss of let-7 expression leads to the modification of temporal regulation of the metamorphosis process (Caygill, 2008). During fly metamorphosis, the expression of let-7 complex (let-7C), a polycistronic locus encoding the let-7, miR-100 and miR-125 miRNAs, is under direct control by the steroid hormone ecdysone. Ecdysone is the central regulator of insect developmental transitions. Therefore, let-7 has been proposed to be part of a conserved, ecdysone regulated pathway that controls the timing of the larva to adult transition (Stratoulias, 2014).

In addition to affecting the metamorphosis clock, Sokol and colleagues have shown that the let-7 deletion also affects the neuromuscular remodeling that takes place during the larva to adult transition (Sokol, 2008). During neuromuscular remodeling, and under normal conditions, the dorsal internal oblique muscles (DIOMs) disappear 12 hours after emergence of the adult fly from the pupa. However, the adult let-7 mutants retain the DIOMs through adulthood. Deletion of the let-7 gene is sufficient to induce this phenotype, while deletion of either miR-100 or miR-125 genes is not enough to recapitulate the DIOM phenotype. Furthermore, let-7 has been shown to govern the maturation of neuromuscular junction of adult abdominal muscles, through regulation of Abrupt expression (Caygill, 2008; Stratoulias, 2014 and references therein).

While previous studies have demonstrated that the let-7 target Abrupt and ecdysone signaling are required for oogenesis in fruit fly ovaries (Jang, 2009), and that the let-7 miRNA family is abundantly expressed both in newborn mouse ovaries and in fly ovaries, no study has been conducted on the role of Lin-28/let-7 network in Drosophila ovaries. Therefore, a study was undertaken of the effects of lin-28 during Drosophila melanogaster development from the egg to the adult, and more particularly during oogenesis (Stratoulias, 2014).

A lin-28 mutant was generated, and the consequent increase of let-7 maturation was validated. lin-28 knockout resulted in reduced muscular performance and defects in DIOM morphogenesis. These results were in line with the let-7 knock out muscular phenotype described earlier (Sokol, 2008). Moreover, this study identified multiple defects during oogenesis due to abnormal follicle and germline stem cell (FSCs and GSCs respectively) differentiation. A link is proposed between ovarian defects and ectopic expression of Fasciclin2 (Fas2), a known downstream target of the Ecdysone pathway, and a predicted let-7 target (Stratoulias, 2014).

Because of their role during stem cell differentiation, members of the let-7 miRNA family have been extensively studied. However, the role of lin-28 is still poorly documented. Deletion of let-7 in Drosophila impairs the musculature remodeling during the larva to adult metamorphosis. For instance the DIOMs, muscles which are required for eclosion and which are lost within 12 hours after eclosion, they are maintained during adulthood upon let-7 deletion (Sokol, 2008). By generating the first lin-28 deletion in flies, this study has successfully confirmed the involvement of Lin-28/let-7 regulatory network in DIOM remodeling. This study has shown that deletion of lin-28 leads to over maturation of let-7, which negatively affects, and sometimes prevents DIOM formation. This drastic phenotype leads to a suboptimal muscular phenotype. However, due to a variable penetrance of the lin-28 deletion phenotype, a proportion of the flies could eclose and live as fertile animals (Stratoulias, 2014).

In addition, a link was discovered between Lin-28 function and oogenesis. The data indicates a role of let-7 during GSC differentiation and egg chamber formation. Because of the importance of these processes, let-7 maturation has to be strictly regulated by Lin-28 activity. It is suggested that a potential network involving Lin-28/let-7/Ecdysone signaling/Abrupt/Fas2 is needed during GSC differentiation and BC migration. The role of Abrupt in downregulating the steroid hormone Ecdysone has previously been demonstrated (Konig, 2011). Indeed, the loss of Taiman, a target of the transcription factor Abrupt and co-activator of Ecdysone receptor, leads to an increase of undifferentiated GSCs in the germarium due to disruption of Ecdysone signaling (Konig. 2011; Jang, 2009). Therefore, by regulating the expression pattern of Abrupt, Lin28/let-7 may adjust the domain of Ecdysone activity, providing a control over the GSCs differentiation and egg chamber maturation during the oogenesis. Indeed, it has been shown that the Ecdysone titre rises during oogenesis at stage 9. While the precise Ecdysone expression pattern is not known, it is suggested that the uniform EcR expression pattern in follicle cells in lin-28 mutants may break the Ecdysone signaling asymmetry needed during proper oogenesis (Stratoulias, 2014).

Furthermore, a previous study demonstrated the activation of let-7 expression via Ecdysone activity (Chawla, 2012). This study showed that lin-28 deletion, resulted in the alleviation of Lin28's inhibitory role on let-7 maturation. This led to loss of Abrupt, which in turn inhibited Ecdysone activity and maintained Fas2 expression, resulting in BC migration impairment. To test whether the increase of Ecdysone signaling amplifies let-7 expression through a positive feedback loop (Chawla, 2012), a system was generated in which there is no control of either let-7 expression nor of Ecdysone activity. This situation leads to an early cyst fusion, a loss of proper GSC differentiation and a mitotic defect, as was observed in the homozygous lin-28dF30 ovaries. The accumulation of these defects may be enough to trigger apoptosis at mid-oogenesis, a well-known checkpoint previously described (Stratoulias, 2014).

Interestingly, the variable penetrance of the phenotype allows proper oogenesis and appearance of subfertile adult flies. This suggests a robust molecular network where feedback loops can rescue the system if one component disturbs the balance (Stratoulias, 2014).

By combining these results with previously published studies, a conserved link is suggested between hormonal signaling and germline stem cell differentiation, involving the let-7 miRNA family. This suggestion is reinforced in the last couple of years by the discovery of dormant ovarian follicles and mitotically active germ cells in adult mammalian ovaries, which are responsive to gonadotropin hormone. Moreover, it has been demonstrated that Lin-28 is involved in germline stem cell regulation in human ovary (Childs, 2012) and in the ovarian surface epithelium of severe ovarian infertility patients axonal projection is critical for assembly of a functional sensory circuit (Stratoulias, 2014).

Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine

Stem cells switch between asymmetric and symmetric division to expand in number as tissues grow during development and in response to environmental changes. The stem cell intrinsic proteins controlling this switch are largely unknown, but one candidate is the Lin-28 pluripotency factor. A conserved RNA-binding protein that is downregulated in most animals as they develop from embryos to adults, Lin-28 persists in populations of adult stem cells. Its function in these cells has not been previously characterized. This study reports that Lin-28 is highly enriched in adult intestinal stem cells in the Drosophila intestine. lin-28 null mutants are homozygous viable but display defects in this population of cells, which fail to undergo a characteristic food-triggered expansion in number and have reduced rates of symmetric division as well as reduced insulin signaling. Immunoprecipitation of Lin-28-bound mRNAs identified Insulin-like Receptor (InR), forced expression of which completely rescues lin-28-associated defects in intestinal stem cell number and division pattern. Furthermore, this stem cell activity of lin-28 is independent of one well-known lin-28 target, the microRNA let-7, which has limited expression in the intestinal epithelium. These results identify Lin-28 as a stem cell intrinsic factor that boosts insulin signaling in intestinal progenitor cells and promotes their symmetric division in response to nutrients, defining a mechanism through which Lin-28 controls the adult stem cell division patterns that underlie tissue homeostasis and regeneration (Chen, 2015).

This study reports that the RNA-binding protein Lin-28 enhances insulin signaling in ISCs and promotes their symmetric renewal independently of let-7. This conclusion is based on observations that Lin-28 is enriched in intestinal progenitor cells; that the founding population of ISCs fail to expand in adult lin-28 mutants; that lin-28 mutant ISCs display reduced rates of symmetric renewal as well as reduced insulin signaling; that Lin-28 physically associates with InR mRNA; and that forced expression of InR rescues lin-28-associated defects in ISC number and symmetric division rates. Building on these results, a model is proposed in which Lin-28 boosts InR levels specifically in progenitor cells during nutrient deprivation. Elevated InR sensitizes ISCs to insulin, poising them to divide symmetrically and thereby driving the expansion of the intestinal epithelium that will maximize nutrient absorption after feeding. More generally, these findings suggest that stem cell competition for insulin, based on cell intrinsic levels of InR, might contribute to the stem cell population dynamics that underlie tissue growth and homeostasis of cycling tissues (Chen, 2015).

Although Lin-28 modulates insulin signaling specifically within progenitor cells, it is not a constitutive component of the IIS pathway. Unlike null alleles in core InR pathway components, lin-28 mutants are viable, proceed through development on schedule, and are of normal size. In addition, lin-28 is dispensable in the intestine for some events known to require InR, such as growth of enterocytes. Furthermore, even in progenitor cells, lin-28 null mutant phenotypes are weaker than those previously described for InR null alleles: InR is required for cell division, whereas lin-28 is required for food-triggered elevations in proliferation and symmetric renewal rates. These observations indicate that some basal level of insulin signaling occurs in the absence of Lin-28 and that Lin-28 boosts insulin signaling in certain cells under certain conditions (Chen, 2015).

An open question relevant to the current model is precisely how Lin-28 affects division pattern. Most simply, enhanced insulin signaling during cell division might promote stem cell identity by increasing the metabolism and/or size of both daughters. However, the possibility cannot be ruled out that Lin-28 might affect cell polarity via, for example, the Par complex, although asymmetric localization of Lin-28 during ISC division has not been detected. Furthermore, Lin-28 activity could also repress Notch signaling, as lower Notch activity leads to ISC expansion. However, such an effect is likely to be indirect because no components of the Notch pathway were found in the Lin-28 immunoprecipitation (Chen, 2015).

Although Lin-28 has been implicated in translational control in vertebrate systems, the precise mechanism remains unknown. The work presented in this study suggests that Lin-28 might directly regulate the translation of the InR mRNA, perhaps via its 5'UTR. Translation of InR mRNA is known to be post-transcriptionally stimulated via its 5'UTR in a cap-independent manner. This probably leads to elevated levels of InR protein in nutrient-deprived cells, which sensitizes them to insulin and thereby ensures a rapid response when growth conditions are restored. Lin-28 might also regulate InR via a 3'UTR mechanism, as it was recently shown that the microRNA miR-305 negatively regulates InR levels in ISCs via its 3'UTR (Chen, 2015).

During C. elegans development, LIN-28 promotes symmetric division of progenitor cells independently of let-7 and related microRNAs. Thus, the well-characterized negative feedback between Lin-28 and let-7 might, in general, be ancillary to their main functions in vivo. Future work will determine whether endogenous Lin-28 promotes the expansion of vertebrate stem cell populations, such as primordial germ cells and neuronal stem cells, by boosting the proportion of stem cells undergoing symmetric renewal (Chen, 2015).


Functions of Lin-28 orthologs in other species

Coupled Caspase and N-end rule ligase activities allow recognition and degradation of pluripotency factor LIN-28 during non-apoptotic development

Recent findings suggest that components of the classical cell death machinery also have important non-cell-death (non-apoptotic) functions in flies, nematodes, and mammals. However, the mechanisms for non-canonical caspase substrate recognition and proteolysis, and the direct roles for caspases in gene expression regulation, remain largely unclear. This study reports that CED-3 caspase and the Arg/N-end rule pathway cooperate to inactivate the LIN-28 pluripotency factor in seam cells, a stem-like cell type in Caenorhabditis elegans, thereby ensuring proper temporal cell fate patterning. Importantly, the caspase and the E3 ligase execute this function in a non-additive manner. CED-3 caspase and the E3 ubiquitin ligase UBR-1 form a complex that couples their in vivo activities, allowing for recognition and rapid degradation of LIN-28 and thus facilitating a switch in developmental programs. The interdependence of these proteolytic activities provides a paradigm for non-apoptotic caspase-mediated protein inactivation (Weaver, 2017).

Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway

The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3' oligo-uridylation activity of TUT4 and TUT7. The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by the exonuclease Dis3l2, a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition this study determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. This study identified three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28-let-7 pathway (Faehnle, 2014).

Lin28 sustains early renal progenitors and induces Wilms tumor

Wilms Tumor, the most common pediatric kidney cancer, evolves from the failure of terminal differentiation of the embryonic kidney. This study shows that overexpression of the heterochronic regulator Lin28 during kidney development in mice markedly expands nephrogenic progenitors by blocking their final wave of differentiation, ultimately resulting in a pathology highly reminiscent of Wilms tumor. Using lineage-specific promoters to target Lin28 to specific cell types, Wilms tumor was observed only when Lin28 is aberrantly expressed in multiple derivatives of the intermediate mesoderm, implicating the cell of origin as a multipotential renal progenitor. Withdrawal of Lin28 expression reverts tumorigenesis and markedly expands the numbers of glomerulus-like structures; tumor formation is suppressed by enforced expression of Let-7 microRNA. Finally, overexpression was demonstrated of the LIN28B paralog in a significant percentage of human Wilms tumor. These data thus implicate the Lin28/Let-7 pathway in kidney development and tumorigenesis (Urbach, 2014).

Lin28 enhances tissue repair by reprogramming cellular metabolism

Regeneration capacity declines with age, but why juvenile organisms show enhanced tissue repair remains unexplained. Lin28a, a highly conserved RNA-binding protein expressed during embryogenesis, plays roles in development, pluripotency, and metabolism. To determine whether Lin28a might influence tissue repair in adults, the reactivation of Lin28a expression was engineered in several models of tissue injury. Lin28a reactivation improved hair regrowth by promoting anagen in hair follicles and accelerated regrowth of cartilage, bone, and mesenchyme after ear and digit injuries. Lin28a inhibits let-7 microRNA biogenesis; however, let-7 repression was necessary but insufficient to enhance repair. Lin28a bound to and enhanced the translation of mRNAs for several metabolic enzymes, thereby increasing glycolysis and oxidative phosphorylation (OxPhos). Lin28a-mediated enhancement of tissue repair was negated by OxPhos inhibition, whereas a pharmacologically induced increase in OxPhos enhanced repair. Thus, Lin28a enhances tissue repair in some adult tissues by reprogramming cellular bioenergetics (Shyh-Chang, 2013).

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. This study uncovered a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. This study shows that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, it is proposed that LIN28 is an essential factor of nucleologenesis during early embryonic development (Vogt, 2012).

LIN28A is a suppressor of ER-associated translation in embryonic stem cells

LIN28 plays a critical role in developmental transition, glucose metabolism, and tumorigenesis. At the molecular level, LIN28 is known to repress maturation of let-7 microRNAs and enhance translation of certain mRNAs. This study obtained a genome-wide view of the molecular function of LIN28A in mouse embryonic stem cells by carrying out RNA crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome footprinting. In addition to let-7 precursors, LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendoplasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins. This study suggests a selective regulatory mechanism for ER-associated translation and reveals an unexpected role of LIN28A as a global suppressor of genes in the secretory pathway (Cho, 2012).

Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms

Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. This study finds that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall these results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy (Piskounova, 2011).

lin-28 controls the succession of cell fate choices via two distinct activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. This study found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, this study finds that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, evidence is provided that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. The evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession (Moss, 2012).

Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites

The heterochronic gene lin-28 is a regulator of developmental timing in the nematode Caenorhabditis elegans. It must be expressed in the first larval stage and downregulated by the second stage for normal development. This downregulation is mediated in part by lin-4, a 21-nt microRNA. If downregulation fails due to a mutation in a short sequence in the lin-28 3' UTR that is complementary to lin-4, then a variety of somatic cell lineages fail to progress normally in development. This study reports that Lin-28 homologues exist in diverse animals, including Drosophila, Xenopus, mouse, and human. These homologues are characterized by the LIN-28 protein's unusual pairing of RNA-binding motifs: a cold shock domain (CSD) and a pair of retroviral-type CCHC zinc knuckles. Conservation of LIN-28 proteins shows them to be distinct from the other conserved family of CSD-containing proteins of animals, the Y-box proteins. Importantly, the LIN-28 proteins of Drosophila, Xenopus, and mouse each appear to be expressed and downregulated during development, consistent with a conserved role for this regulator of developmental timing. In addition, the extremely long 3' UTRs of mouse and human Lin-28 genes show extensive regions of sequence identity that contain sites complementary to the mammalian homologues of C. elegans lin-4 and let-7 microRNAs, suggesting that microRNA regulation is a conserved feature of the Lin-28 gene in diverse animals (Moss, 2003).

LIN28 zinc knuckle domain is required and sufficient to induce let-7 oligouridylation

LIN28 (see Drosophila Lin28) is an RNA binding protein that plays crucial roles in pluripotency, glucose metabolism, tissue regeneration, and tumorigenesis. LIN28 binds to the let-7 (see Drosophila let-7) primary and precursor microRNAs through bipartite recognition and induces degradation of let-7 precursors (pre-let-7) by promoting oligouridylation by terminal uridylyltransferases (TUTases; see Drosophila Tailor). This study report that the zinc knuckle domain (ZKD) of mouse LIN28 recruits TUT4 to initiate the oligouridylation of let-7 precursors. Crystal structure of human LIN28 in complex with a fragment of pre-let-7f-1 determined to 2.0 Å resolution shows that the interaction between ZKD and RNA is constrained to a small cavity with a high druggability score. The specific interaction between ZKD and pre-let-7 was shown to be necessary and sufficient to induce oligouridylation by recruiting the N-terminal fragment of TUT4 (NTUT4) and the formation of a stable ZKD:NTUT4:pre-let-7 ternary complex is crucial for the acquired processivity of TUT4 (Wang, 2017).


REFERENCES

Search PubMed for articles about Drosophila Lin-28

Ambros, V. and Horvitz, H. R. (1984). Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226: 409-416. PubMed ID: 6494891

Caygill, E. E. and Johnston, L. A. (2008). Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs. Curr Biol 18: 943-950. PubMed ID: 18571409

Chawla, G. and Sokol, N. S. (2012). Hormonal activation of let-7-C microRNAs via EcR is required for adult Drosophila melanogaster morphology and function. Development 139: 1788-1797. PubMed ID: 22510985

Chen, C. H., Luhur, A. and Sokol, N. (2015). Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine. Development 142: 3478-3487. PubMed ID: 26487778

Childs, A. J., Kinnell, H. L., He, J. and Anderson, R. A. (2012). LIN28 is selectively expressed by primordial and pre-meiotic germ cells in the human fetal ovary. Stem Cells Dev 21: 2343-2349. PubMed ID: 22296229

Cho, J., Chang, H., Kwon, S. C., Kim, B., Kim, Y., Choe, J., Ha, M., Kim, Y. K. and Kim, V. N. (2012). LIN28A is a suppressor of ER-associated translation in embryonic stem cells. Cell 151: 765-777. PubMed ID: 23102813

Faehnle, C. R., Walleshauser, J. and Joshua-Tor, L. (2014). Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway. Nature 514: 252-256. PubMed ID: 25119025

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811. PubMed ID: 9486653

Jang, A. C., Chang, Y. C., Bai, J. and Montell, D. (2009). Border-cell migration requires integration of spatial and temporal signals by the BTB protein Abrupt. Nat Cell Biol 11: 569-579. PubMed ID: 19350016

Jin, J., Jing, W., Lei, X. X., Feng, C., Peng, S., Boris-Lawrie, K. and Huang, Y. (2011). Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes. Nucleic Acids Res 39: 3724-3734. PubMed ID: 21247876

Konig, A., Yatsenko, A. S., Weiss, M. and Shcherbata, H. R. (2011). Ecdysteroids affect Drosophila ovarian stem cell niche formation and early germline differentiation. EMBO J 30: 1549-1562. PubMed ID: 21423150

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Biological Overview

date revised: 20 December 2014

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