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Open Access Publications from the University of California

Scholarly Works (31 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2018)
In this issue of Molecular Cell, Ivanov et al. (2018) demonstrate how cells sense metabolite levels and regulate production of their biosynthetic enzymes through the formation of "ribosome queues."
    Cover page: The Traffic Jam: Polyamine Prevalence Pauses Protein Production
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2016)
    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5' and 3' untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5' untranslated regions exert robust translational control between cell lines, while 3' untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.
      • Thesis
      • Peer Reviewed
      UC San Francisco Electronic Theses and Dissertations (2022)

      Maintaining protein homeostasis is essential for a proper functioning cell. From the time a polypeptide chain begins to be synthesized to the time the final product arrives at its destination there is an incredible amount of regulation taking place. The dysregulation of these processes can have disastrous effects for the cell. In this thesis I will be exploring two important areas of regulation: translation initiation and protein targeting. In Chapter 1, I will show structural, biochemical, and cell-based data characterizing the AAA ATPase ATAD1. ATAD1 is tasked with removing mistargeted tail-anchored proteins from the mitochondrial outer membrane. Through cryo-electron microscopy, we solved the structure of this protein with a peptide trapped in the central pore and designed a cell-based assay to validate structure-based predictions. We identified conserved structural elements that are essential for the function of ATAD1, in particular, the unusually aromatic nature of the pore loops that enable ATAD1 to extract it’s hydrophobic substrates.

      In Chapter 2, I will be describing our characterization of an alternative translation initiation factor eukaryotic initiation factor 2A (eIF2A). From the initial discovery of this protein in the 1970’s, eIF2A has been implicated in non-canonical translation initiation processes and has been shown to be important for responding to stress. Using available genome-wide genetic interaction datasets for Saccharomyces cerevisiae, we identified and characterized the genetic interaction between eIF2A and TIF1 (the DEAD-box helicase component of the eIF4F complex, eIF4A). We found that strains with both proteins deleted exhibited translational reprogramming that resulted in a sensitivity to metabolic stressors. As an exciting entry into detailed in vitro characterization, we also overcame a hurtle in the recombinant expression of the human version of eIF2A and have begun re-examining the previously held assumptions about the mechanism of eIF2A-dependent translation.

        Cover page: Mechanistic investigation of protein homeostasis by two key factors, ATAD1 and eIF2A
        • Thesis
        • Peer Reviewed
        UC San Francisco Electronic Theses and Dissertations (2011)

        The lifetime of eukaryotic messenger RNA is highly regulated by elements on both the 5' and 3' ends of the message. Methylated guanine "cap" on the 5' end of a message is critical for nuclear export, protein translation, and stability of a message. The cap structure is so crucial that many viruses hijack cellular caps to direct translation towards nefarious ends. Removal of the cap structure of a mRNA is a highly regulated, irreversible step that sentences a mRNA to destruction by conserved exonucleases, and is the ultimate goal of many mRNA surveillance and degradation pathways. An enzyme known as Dcp2 executes decapping, or removal of the cap. Dcp2 from yeast to humans contains two conserved domains: one at the very N-terminus of the protein known as the regulatory domain, and a second following directly in sequence of the Nudix hydrolase family. While the catalytic domain contains the core Nudix motif necessary and sufficient for removal of mRNA caps, the regulatory domain binds the partner Dcp1 and is required for decapping in vivo. The dynamics, structure, and functional consequences of interactions between these two domains are the focus of this manuscript. In sum, regulation of decapping appears to occur by modulating the interaction between these two domains, which creates or inhibits formation of a composite active site that apposes absolutely conserved and catalytically essential regions on both domains.

          Cover page: Conformational control of eukaryotic mRNA decapping by Dcp2
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2016)
          Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions.
            Cover page: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2021)
            DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in the context of mRNA translation in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.
              Cover page: DDX3X and DDX3Y are redundant in protein synthesis
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2010)
              Decapping by Dcp2 is an essential step in 5'-to-3' mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step by two orders of magnitude and block the formation of the closed state, whereas Dcp1 enhances the catalytic step by a factor of 10 and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping where coactivators trigger decapping by stabilizing a labile composite active site.
                Cover page: A split active site couples cap recognition by Dcp2 to activation
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2019)
                Polypyrimidine tract-binding proteins (PTBPs) are RNA binding proteins that regulate a number of posttranscriptional events. Human PTBP1 transits between the nucleus and cytoplasm and is thought to regulate RNA processes in both. However, information about PTBP1 mRNA isoforms and regulation of PTPB1 expression remains incomplete. Here we mapped the major PTBP1 mRNA isoforms in HEK293T cells and identified alternative 5' and 3' untranslated regions (5'-UTRs, 3'-UTRs), as well as alternative splicing patterns in the protein coding region. We also assessed how the observed PTBP1 mRNA isoforms contribute to PTBP1 expression in different phases of the cell cycle. Previously, PTBP1 mRNAs were shown to crosslink to eukaryotic translation initiation factor 3 (eIF3). We find that eIF3 binds differently to each PTBP1 mRNA isoform in a cell cycle dependent manner. We also observe a strong correlation between eIF3 binding to PTBP1 mRNAs and repression of PTBP1 levels during the S phase of the cell cycle. Our results provide evidence of translational regulation of PTBP1 protein levels during the cell cycle, which may affect downstream regulation of alternative splicing and translation mediated by PTBP1 protein isoforms.
                  Cover page: PTBP1 mRNA isoforms and regulation of their translation
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2013)
                  Removal of the 5' cap structure by Dcp2 is a major step in several 5'-3' mRNA decay pathways. The activity of Dcp2 is enhanced by Dcp1 and bound coactivators, yet the details of how these interactions are linked to chemistry are poorly understood. Here, we report three crystal structures of the catalytic Nudix hydrolase domain of Dcp2 that demonstrate binding of a catalytically essential metal ion, and enzyme kinetics are used to identify several key active site residues involved in acid/base chemistry of decapping. Using nuclear magnetic resonance and molecular dynamics, we find that a conserved metal binding loop on the catalytic domain undergoes conformational changes during the catalytic cycle. These findings describe key events during the chemical step of decapping, suggest local active site conformational changes are important for activity, and provide a framework to explain stimulation of catalysis by the regulatory domain of Dcp2 and associated coactivators.
                    Cover page: Active Site Conformational Dynamics Are Coupled to Catalysis in the mRNA Decapping Enzyme Dcp2
                    • Article
                    • Peer Reviewed
                    UC Berkeley Previously Published Works (2017)
                    Faithful cellular differentiation requires temporally precise activation of gene expression programs, which are coordinated at the transcriptional and translational levels. Neurons express the most complex set of mRNAs of any human tissue, but translational changes during neuronal differentiation remain incompletely understood. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and measured genome-wide RNA and translation levels with transcript-isoform resolution. We found that thousands of genes change translation status during differentiation without a corresponding change in RNA level. Specifically, we identified mTOR signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by regulatory sequences in 3' UTRs. Together, our findings identify extensive translational control changes during human neuronal differentiation and a crucial role of 3' UTRs in driving cell-type-specific translation.
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