Piwi-interacting (pi) RNAs are germline-expressed small RNAs linked to epigenetic programming. C. elegans piRNAs are thought to be transcribed as independent gene-like loci. To test this idea and to ...identify potential transcription start (TS) sites for piRNA precursors, we developed CapSeq, an efficient enzymatic method for 5′ anchored RNA profiling. Using CapSeq, we identify candidate TS sites, defined by 70–90 nt sequence tags, for >50% of annotated Pol II loci. Surprisingly, however, these CapSeq tags failed to identify the overwhelming majority of piRNA loci. Instead, we show that the likely piRNA precursors are ∼26 nt capped small (cs) RNAs that initiate precisely 2 nt upstream of mature piRNAs and that piRNA processing or stability requires a U at the csRNA +3 position. Finally, we identify a heretofore unrecognized class of piRNAs processed from csRNAs that are expressed at promoters genome wide, nearly doubling the number of piRNAs available for genome surveillance.
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► A new enzymatic method enriches 5′ ends of Pol II transcripts ► Identification of transcription start sites for most C. elegans Pol II genes ► Capped small RNAs of ∼26 nt are expressed from Pol II promoters genome wide ► csRNAs, but not longer capped RNAs, are the piRNA precursors in C. elegans
A new method for genome-wide cloning of RNA polymerase II transcripts reveals capped 26-nucleotide RNAs as the elusive precursors for C. elegans piRNAs, which silence foreign nucleic acids.
Non‐stop mRNAs, which lack a stop codon due to mutation or errors in mRNA transcription or processing, serve as templates for production of aberrant and potentially toxic protein products. ...Ultimately, translation of non‐stop mRNAs leads to a failure in normal translation termination and ribosome collisions, which in eukaryotes initiates ubiquitin‐dependent degradation of the protein product and Non‐Stop Decay (NSD) of the problematic mRNA. Ribosome collisions in yeast are linked to activation of cellular stress responses and failure to resolve stalled ribosomes leads to neurodegeneration in mice, highlighting the biological importance of mRNA quality control.
In the nematode Caenorhabditis elegans, loss of the core NSD components pelo‐1 and skih‐2 leads to temperature‐sensitive sterility, as does the loss of endogenous siRNAs that are triggered by NSD at some loci. To further probe the link between NSD and the biology of germ cells, we have used an RNAi approach to screen for temperature‐sensitive reduction in brood size for the C. elegans orthologs of several proteins known to be part of the NSD machinery in other organisms. Preliminary results have identified 2 NSD‐associated genes with conditionally reduced brood sizes. We will present progress on examining the requirement of these genes for Non‐Stop decay in C. elegans, as well as investigation of their specific contributions to the biology of the germ line.
Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic ...approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs.
Peptide release, the reaction that hydrolyzes a completed protein from the peptidyl-tRNA upon completion of translation, is catalyzed in the active site of the large subunit of the ribosome and ...requires a class I release factor protein. The ribosome and release factor protein cooperate to accomplish two tasks: recognition of the stop codon and catalysis of peptidyl-tRNA hydrolysis. Although many fundamental questions remain, substantial progress has been made in the past several years. This review summarizes those advances and presents current models for the mechanisms of stop codon specificity and catalysis of peptide release. Finally, we discuss how these views fit into a larger emerging theme in the translation field: the importance of induced fit and conformational changes for progression through the translation cycle.
Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi, and nematodes, cellular RNA-dependent RNA ...polymerases (RdRPs) use AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in Caenorhabditis elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4 are required for the biogenesis of 26-nt small RNAs with a 5' guanine (26G-RNAs) and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a nonrandom distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control overexpression resulting from gene expansion.
Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and ...aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.
In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first ...microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.
Peptide release on the ribosome is catalyzed in the large subunit peptidyl transferase center by release factors on recognition of stop codons in the small subunit decoding center. Here we examine ...the role of the decoding center in this process. Mutation of decoding center nucleotides or removal of 2′OH groups from the codon—deleterious in the related process of tRNA selection—has only mild effects on peptide release. The miscoding antibiotic paromomycin, which binds the decoding center and promotes the critical steps of tRNA selection, instead dramatically inhibits peptide release. Differences in the kinetic mechanism of paromomycin inhibition on stop and sense codons, paired with correlated structural changes monitored by chemical footprinting, suggest that recognition of stop codons by release factors induces specific structural rearrangements in the small subunit decoding center. We propose that, like other steps in translation, the specificity of peptide release is achieved through an induced-fit mechanism.
As it has become increasingly clear that the RNA components of the ribosome are central to its function, the in vitro analysis of mutations in the ribosomal RNAs has become an important tool for ...understanding the molecular details of ribosome function. However, the frequent dominant lethal phenotypes of mutations at interesting rRNA residues has long presented a hurdle to this analysis, as their lethality has rendered it impossible to generate pure populations of in vivo-derived ribosomes for study. We present here the details of a method for affinity purification of ribosomes bearing any mutation in the 16S or 23S rRNA and demonstrate that ribosomes purified using this technology are highly active in the several steps of translation we have examined.
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