From early lessons to new frontiers: the worm as a treasure trove of small RNA biology.

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.

CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors.

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.

Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway.

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.

Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline.

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.

Peptide release on the ribosome depends critically on the 2' OH of the peptidyl-tRNA substrate.

Peptide release on the ribosome is catalyzed by protein release factors (RFs) on recognition of stop codons positioned in the A site of the small ribosomal subunit. Here we show that the 2' OH of the peptidyl-tRNA substrate plays an essential role in catalysis of the peptide release reaction. These observations parallel earlier studies of the mechanism of the peptidyl transfer reaction and argue that related mechanisms are at the heart of catalysis for these reactions.

Peptide release on the ribosome: mechanism and implications for translational control.

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.

Stop codon recognition by release factors induces structural rearrangement of the ribosomal decoding center that is productive for peptide release.

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.

Ribosomal translocation: LepA does it backwards.

During translation, mRNA is threaded through the ribosome in precise and directional three-nucleotide steps. A recent paper identifies a new GTPase, LepA, which catalyzes unexpected one-codon backward movement on the ribosome.

The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity.

Ribosomal variants carrying mutations in active site nucleotides are severely compromised in their ability to catalyze peptide bond formation (PT) with minimal aminoacyl tRNA substrates such as puromycin. However, catalysis of PT by these same ribosomes with intact aminoacyl tRNA substrates is uncompromised. These data suggest that these active site nucleotides play an important role in the positioning of minimal aminoacyl tRNA substrates but are not essential for catalysis per se when aminoacyl tRNAs are positioned by more remote interactions with the ribosome.

Affinity purification of in vivo-assembled ribosomes for in vitro biochemical analysis.

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.

The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release.

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.