RNA molecules are diverse in form and function. They include messenger RNAs (mRNAs) that are templates for proteins, splice products such as introns that can generate functional noncoding RNAs, and a slew of smaller RNAs such as transfer RNAs (tRNAs) that help decode mRNAs into proteins. RNAs can show distinct patterns of subcellular localization that play an important role in protein localization. However, RNA distribution in cells is incompletely understood, with prior studies focusing primarily on RNAs that are long (>200 nucleotides), fully processed, and polyadenylated. We examined the distribution of RNAs in neurons. Neuronal compartments can be separated by long distances and play distinct roles, raising the possibility that RNA localization is especially overt and functionally meaningful in these cells. In our exploration, we physically dissected projections from cell bodies of neurons from the rat brain and sequenced total RNA. We describe two main findings. First, we identified excised introns that are enriched in neuronal projections and confirmed their localization by single- molecule fluorescence in situ hybridization. These are a previously unknown set of circular RNAs in neuronal projections: tailless lariats that possess a non- canonical C branchpoint. Second, we observed a highly abundant population of small (20-150 nucleotide) RNAs in neuronal projections, most of which are tRNAs. For both circular introns and tRNAs, we did not observe known RNA localization signals. Thus, many types of RNA, if sufficiently stable, appear free to diffuse to distant locations, their localization perhaps aided by the movement of large organelles in the confines of neuronal projections. Our survey of RNA molecules across subcellular compartments provides a foundation for investigating the function of these molecules and the mechanisms that localize them.

Ancestral environmental conditions can instruct offspring development, although the mechanism(s) underlying such transgenerational epigenetic inheritance is unclear. In murine models focused on paternal dietary effects, we and others have identified tRNA fragments (tRFs) in mature sperm as potential carriers of epigenetic information. In our search for molecular targets of specific tRFs, we observed that altering the level of 5’-tRF Glycine-GCC (tRF-GG) in mouse embryonic stem cells (mESCs) and preimplantation embryos modulates the expression of the endogenous retrovirus MERV-L and genes regulated by MERV-L. Intriguingly, transient derepression of MERV-L is associated with totipotency of two-cell stage embryos and a subset of two-cell-like mESCs. Here, I reveal the mechanistic basis for tRF-GG regulation of MERV-L. I show that tRF-GG supports the production of numerous small nuclear RNAs associated with the Cajal body, in mouse and human embryonic stem cells. In particular, tRF-GG modulates the levels of U7 snRNA to ensure an adequate supply of histone proteins. This in turn safeguards heterochromatin-mediated transcriptional repression of MERV-L elements. Importantly, tRF-GG effects on histone mRNA levels, activity of a histone 3’UTR reporter, and expression of MERV-L associated transcripts can all be suppressed by appropriate manipulation of U7 RNA levels. I also show that hnRNPF and H bind directly to tRF-GG, and display a stark overlap of in vivo functions to tRF-GG. Together, this data uncovers a conserved mechanism for a 5’ tRNA fragment in the fine-tuning of a regulatory cascade to modulate global chromatin organization during pre-implantation development.

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There is strong crystallographic evidence that the 23S rRNA is the only catalytic entity in the peptidyl transferase center. Various mechanisms for the catalysis of peptidyl transfer have been proposed. Recently, attention has been given to the idea that the 23S rRNA simply acts to position the tRNA for spontaneous peptidyl transfer and that chemical catalysis may play only a secondary role. Conserved nucleotides U2585 and U2506 are thought to be involved in positioning the 3’ ends of A- and P-site substrates based on the crystallographic evidence, and because mutagenesis at these sites severely impairs peptide bond formation. In this study, pure populations of ribosomes with either U2585A or U2506G mutations in the 23S rRNA were analyzed to test the hypothesis that substitutions at nucleotides U2585 and U2506 in the peptidyl transferase center impair peptide bond formation by altering the position of the 3’ end of P-site tRNA relative to the 23S rRNA. Pure populations of mutant or wild-type ribosomes were obtained by an affinity tagging system and probed with 32P-labeled [2N3A76]tRNAPhe to determine how the 3’ end of tRNA interacts with the ribosomal proteins and 23S RNA at the peptidyl transferase center. Some of the data for the ribosomes with a G at position 2506 are consistent with a model suggested by Schmeing and coworkers in which nucleotide U2506 breaks from its original wobble base pair with nucleotide G2583 during A-site tRNA binding and swings towards the 3’ end of P-site tRNA, while nucleotide U2585 simultaneously moves away from the 3’ end of P-site tRNA.

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No description available.

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tRNA genes are highly transcribed and perform one of the most fundamental cellular functions. Although a universal pattern observed across all three domains of life is that highly transcribed genes tend to evolve slowly, tRNA genes have been shown previously to evolve rapidly. This rapid sequence evolution could result from relaxed selection, increased mutation rate, or a combination of both. Here, we use mutation-accumulation line sequencing data to show that tRNA genes accumulate more mutations than other gene types. Our results indicate that this elevated mutation rate is a consequence of both elevated transcription-associated mutagenesis and a lack of transcription-coupled repair in tRNA genes. We also identify the gene MSH2 as being involved in transcription-coupled repair.