This year over 350 Bay Area RNA researchers convened on the UCSF campus to mingle and schmooze, have a coffee, and discuss all things related to RNA. The meeting comprised 19 graduate student and postdoc talks and more than 30 posters. Out of these excellent presentations Courtney French (Doudna Lab, UC Berkeley) and Shifeng Xue (Barna Lab, UCSF), and Shelley Starck (Shastri Lab, UC Berkeley) won awards.
Poster Award- Courtney French (graduate student, Brenner Lab)
Courtney is a second year graduate student in Steven Brenner's group at UC Berkeley (MCB dept). She has an MA in Biotechnology from Boston University and grew interested in computational biology while working as a research technician at the Broad Institute. She is currently using RNA-seq analysis to explore mRNA splicing and degradation in diverse species. Her abstract is below.
Almost two thousand genes are targeted by nonsense-mediated mRNA decay in a human cell line. Many alternatively spliced isoforms contain a premature termination codon that targets them for degradation by the nonsense-mediated mRNA decay (NMD) surveillance system. Some such unproductive splicing events have a regulatory function, where alternative splicing and NMD act together to impact protein expression. To effectively survey the targets of NMD genome-wide, we performed RNA-Seq on human HeLa cells in which NMD had been inhibited via knockdown of UPF1. By comparing the transcriptomes of cells where NMD has been inhibited to those of cells where NMD remained active, we found thousands of alternatively spliced isoforms that are potentially degraded by NMD. We employed the canonical 50nt rule to find isoforms with a premature termination codon and identified those that had a significant 1.5x increase in abundance when NMD is inhibited as putative NMD targets, resulting in a conservative set of 2,443 isoforms corresponding to 1,924 genes (18% of expressed genes). Our results verify the activity of NMD on previously inferred unproductive isoforms for the SR proteins and hnRNPs, and indicate that alternative splicing coupled with NMD regulates many more splicing factor genes. Additionally, we discovered thousands of new NMD-targeted mRNAs from genes involved a broad range of functional processes. Finally, we characterized the effect of different isoform features on the likelihood of NMD degradation. We saw that while longer 3’-UTRs result in a slight increase in NMD (up to 40% of isoforms are degraded), the canonical 50nt rule has a much stronger effect (>70% of isoforms are degraded). Our findings demonstrate that gene expression regulation through NMD is more widespread than previously inferred, impacting a large number of functionally distinct proteins and processes.
Poster Award- Shifeng Xue (graduate student, Barna Lab)
Shifeng is a third year graduate student at University of California, San Francisco. She grew up in Singapore and got her undergraduate degree from UCSD. Currently she is interested in studying translational control in embryonic development. Her abstract is below.
Specialized ribosome activity in control of gene expression and vertebrate embryonic development. Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory control in gene expression. Through an unbiased forward genetic screen we uncovered that RPL38, a core ribosomal protein, is required to establish the mammalian body plan. In Rpl38 mutant embryos, global protein synthesis is unchanged however transcript-specific translational control of subsets of Homeobox (Hox) mRNAs required for anteroposterior tissue patterning is perturbed. Our data further reveal that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes are observed. By carrying out the first mammalian ribosomal protein (RP) expression screen we extend these findings by uncovering dramatic RP heterogeneity in a cell and tissue specific manner. This led us to propose that ribosomes in distinct regions of the embryo may be highly “specialized”. To date, the regulatory elements in mRNAs that may be recognized be specialized ribosomes remain unknown. Surprisingly, we functionally determined that a common feature of RPL38 target Hox mRNAs is the presence of internal ribosome entry site (IRES) elements that promote translation initiation in a cap-independent manner via RPL38. We propose a model in which upstream open reading frames (uORFs) that we have also identified in these mRNAs maintain cap-dependent translation at a low level thus providing a means to “specialize” translation control through an IRES-dependent mechanism. Our ongoing work seeks to determine whether Hox IRES-elements promote temporal and spatial control of protein production as a new paradigm for regulation of gene expression by specialized ribosome activity.
Poster Award- Shelley Starck (postdoc, Shastri Lab)
Shelley is a senior postdoctoral fellow in Nilabh Shastri’s lab in the MCB Division of Immunology and Pathogenesis at the University of California, Berkeley. She became intrigued with unconventional protein synthesis mechanisms while earning a Ph.D. in Chemistry at the California Institute of Technology and a B.S. degree in Chemistry at the University of Virginia. Her abstract is below.
Initiation with Leu-tRNA shapes the peptide-MHC class I repertoire. An essential function of cellular translation is to supply major histocompatibility complex (MHC) class I molecules with peptides for activation of immune surveillance pathways. These newly synthesized peptides are encoded in conventional AUG-initiated as well as in cryptic, non-AUG initiated reading frames of cellular and viral mRNAs. Whether cells use the same or distinct machinery to translate cryptic peptides at non-AUG start codons, such as CUG, is not known. Here, we show that initiation of peptide precursors at cryptic CUG start codons is regulated by a mechanism distinct from the AUG/Met-tRNAiMet pathway. To determine the mechanism by which CUG diverges from AUG initiation, we isolated CUG initiation complexes, including complexes on the endogenous CUG-initiated Myc isoform. We find they contain a Leu-tRNA, thought to function only during the elongation stage of protein synthesis, which is distinct from initiator Met-tRNAiMet and enhances initiation of antigenic precursors. This CUG/leucine initiation event proceeds independent of Met-tRNAiMet-based initiation, but by an eIF2A-dependent process. Thus, a Leu-initiator tRNA-based mechanism supplies peptides for presentation by MHC class I molecules and could regulate non-AUG-initiated global protein synthesis.
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