Shih-Peng Chan

microRNA-Mediated Silencing Inside P-Bodies

Cytoplasmic processing bodies, or P bodies, contain a high concentration of enzymes and factors required for mRNA turnover and translational repression. Recent studies provide evidence that mRNAs silenced by miRNAs are localized to P bodies for storage or degradation, perhaps in adjacent subcompartments. mRNP remodeling, induced potentially by miRISC or RNA helicase activity, may cause the replacement of the translation initiation complex at the 5’ end of mRNA, translational repression, and localization to P bodies. Further remodeling may facilitate access of the decapping complex to the cap structure. However, with appropriate signals, stored mRNAs in P bodies could be released and returned to the translational machinery through a mechanism that may require binding of regulatory proteins to the 3’ UTR of selected mRNAs.

Analysis of microRNA Expression and Function

Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded within the genome and are initially transcribed as primary transcripts that can be several kilobases in length. Primary transcripts are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce ∼70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs, respectively. Mature miRNAs regulate gene expression post-transcriptionally by imperfectly binding target mRNAs in association with the multiprotein RNA induced silencing complex (RISC). The conserved sequence, expression pattern, and function of some miRNAs across distinct species as well as the importance of specific miRNAs in many biological pathways have led to an explosion in the study of miRNA biogenesis, miRNA target identification, and miRNA target regulation. Many advances in our understanding of miRNA biology have come from studies in the powerful model organism C. elegans. This chapter reviews the current methods used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-protein complexes, and miRNA target regulation.

A Genetic Cascade of let-7-ncl-1-fib-1 Modulates Nucleolar Size and rRNA Pool in Caenorhabditis elegans

Ribosome biogenesis takes place in the nucleolus, the size of which is often coordinated with cell growth and development. However, how metazoans control nucleolar size remains largely unknown. Caenorhabditis elegans provides a good model to address this question owing to distinct tissue distribution of nucleolar sizes and a mutant, ncl-1, which exhibits larger nucleoli than wild-type worms. Here, through a series of loss-of-function analyses, we report that the nucleolar size is regulated by a circuitry composed of microRNA let-7, translation repressor NCL-1, and a major nucleolar pre-rRNA processing protein FIB-1/fibrillarin. In cooperation with RNA binding proteins PUF and NOS, NCL-1 suppressed the translation of FIB-1/fibrillarin, while let-7 targeted the 3’UTR of ncl-1 and inhibited its expression. Consequently, the abundance of FIB-1 is tightly controlled and correlated with the nucleolar size. Together, our findings highlight a novel genetic cascade by which post-transcriptional regulators interplay in developmental control of nucleolar size and function.

RACK-1 regulates let-7 microRNA expression and terminal cell differentiation in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates cell cycle exit and terminal differentiation in the C. elegans heterochronic gene pathway. Low expression of let-7 results in retarded vulva and hypodermal cell development in C. elegans and has been associated with several human cancers. Previously, the versatile scaffold protein receptor for activated C kinase 1 (RACK1) was proposed to facilitate recruitment of the miRNA-induced silencing complex (miRISC) to the polysome and to be required for miRNA function in C. elegans and humans. Here, we show that depletion of C. elegans RACK-1 by RNAi increases let-7 miRNA levels and suppresses the retarded terminal differentiation of lateral hypodermal seam cells in mutants carrying the hypomorphic let-7(n2853) allele or lacking the let-7 family miRNA genes mir-48 and mir-241. Depletion of RACK-1 also increases the levels of precursor let-7 miRNA. When Dicer is knocked down and pre-miRNA processing is inhibited, depletion of RACK-1 still leads to increased levels of pre-let-7, suggesting that RACK-1 affects a biogenesis mechanism upstream of Dicer. No changes in the activity of the let-7 promoter or the levels of primary let-7 miRNA are associated with depletion of RACK-1, suggesting that RACK-1 affects let-7 miRNA biogenesis at the post-transcriptional level. Interestingly, rack-1 knockdown also increases the levels of a few other precursor miRNAs. Our results reveal that RACK-1 controls the biogenesis of a subset of miRNAs, including let-7, and in this way plays a role in the heterochronic gene pathway during C. elegans development.

Genetic control of nucleolar size: An evolutionary perspective.

ABSTRACT Exploiting a C. elegans mutant (ncl-1) exhibiting nucleolar abnormalities, we recently identified the let-7/ncl-1/fib-1 genetic cascade underlying proper rRNA abundance and nucleolar size. These 3 factors, let-7 (a miRNA), NCL-1 (a member of the TRIM-NHL family), and fibrillarin (a nucleolar methyltransferase), are evolutionarily conserved across metazoans. In this article, we provide several lines of bioinformatic evidence showing that human and Drosophila homologues of C. elegans NCL-1, TRIM-71 and Brat, respectively, likely act as translational suppressors of fibrillarin. Moreover, since their 3′-UTRs contain putative target sites, they may also be under the control of the let-7 miRNA. We hypothesize that let-7, TRIM and fibrillarin contribute activities in concert, and constitute a conserved network controlling nucleolar size in eukaryotes. We provide an in-depth literature review of various molecular pathways, including the let-7/ncl-1/fib-1 genetic cascade, implicated in the regulation of nucleolar size.

A novel function for the DEAD-box RNA helicase DDX-23 in primary microRNA processing in Caenorhabditis elegans.

Primary microRNAs (pri-miRNAs) are cleaved by the nuclear RNase III Drosha to produce hairpin-shaped precursor miRNAs (pre-miRNAs). In humans, this process is known to be facilitated by the DEAD-box helicases p68 (DDX5) and p72 (DDX17). In this study, we performed a candidate-based RNAi screen in C. elegans to identify DEAD/H-box proteins involved in miRNA biogenesis. In a let-7(mg279) sensitized genetic background, knockdown of a homolog of yeast splicing factor Prp28p, DDX-23, or a homolog of human helicases p68 and p72, DDX-17, enhanced let-7 loss-of-function phenotypes, suggesting that these helicases play a role in let-7 processing and/or function. In both ddx-23(RNAi) and ddx-17(RNAi), levels of mature let-7 were decreased while pri-let-7 was found to accumulate, indicating that the helicases likely act at the level of pri-let-7 processing. DDX-23 and DDX-17 were also required for the biogenesis of other known heterochronic miRNAs, including lin-4 and the let-7 family members miR-48, miR-84 and miR-241. Their function was not confined to the heterochronic pathway, however, since they were both necessary for down-regulation of cog-1 by the spatial patterning miRNA, lsy-6. Here, we present a novel function for C. elegans DDX-23 in pri-miRNA processing, and also suggest a conserved role for DDX-17 in this process.

BCAS2 Regulates Delta-Notch Signaling Activity through Delta Pre-mRNA Splicing in Drosophila Wing Development

Previously, we showed that BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. In this study, we provide strong evidence that BCAS2 regulates the activity of Delta-Notch signaling via Delta pre-mRNA splicing. Depletion of dBCAS2 reduces Delta mRNA expression and leads to accumulation of Delta pre-mRNA, resulting in diminished transcriptions of Delta-Notch signaling target genes, such as cut and E(spl)m8. Furthermore, ectopic expression of human BCAS2 (hBCAS2) and Drosophila BCAS2 (dBCAS2) in a dBCAS2-deprived fly can rescue dBCAS2 depletion-induced wing damage to the normal phenotypes. These rescued phenotypes are correlated with the restoration of Delta pre-mRNA splicing, which affects Delta-Notch signaling activity. Additionally, overexpression of Delta can rescue the wing deformation by deprivation of dBCAS2; and the depletion of dBCAS2 can restore the aberrant eye associated with Delta-overexpressing retinas; providing supporting evidence for the regulation of Delta-Notch signaling by dBCAS2. Taken together, dBCAS2 participates in Delta pre-mRNA splicing that affects the regulation of Delta-Notch signaling in Drosophila wing development.

dBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.