Biology subjects

Tollervey, D.

Publications and source records attributed to Tollervey, D..

24 records · Page 2Linked to original sources

bioRxiv2020

Stress-induced translation inhibition through rapid displacement of scanning initiation factors

Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome, without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA-association. Among translation components, RNA-association was most reduced for initiation factors involved in 40S scanning (eIF4A, eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5'-ends of mRNAs. Following glucose withdrawal, 5-binding was abolished within 30sec, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5 RNA-binding by initiation factors over [~]16min. Translation shutoff provoked selective 5'-degradation of mRNAs encoding translation-related factors, mediated by Xrn1. These results reveal mechanisms underlying translational control of gene expression during stress. HighlightsA quantitative proteomic approach reveals rapid stress-induced changes in RNA-binding Translation shutdown is driven by loss of mRNA binding by scanning initiation factors eIF4B and Ded1 have key but separate roles in driving the stress response Heat shock invokes rapid RNA degradation by Xrn1, selective for translation machinery

cell biology↗

bioRxiv2020

Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduced Mtr4 recruitment and increased RNA abundance for mRNAs specifically showing high Trf5 binding.

molecular biology↗

bioRxiv2020

Nascent transcript folding plays a major role in determining RNA polymerase elongation rates

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5 bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C-content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/969709v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@f3f9fcorg.highwire.dtl.DTLVardef@1a133c8org.highwire.dtl.DTLVardef@4eae0dorg.highwire.dtl.DTLVardef@18502b8_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSStructures in the nascent RNA correlate with rapid elongation by RNAPI in vivo Stable RNA structures limit RNAPI backtracking in vitro GC content in the transcription bubble tunes transcription elongation rate Nascent transcript folding modulates dynamics of all three RNAPs in vivo

molecular biology↗

bioRxiv2018

Identification of RNA-associated peptides, iRAP, defines precise sites of protein-RNA interaction

ABSRACTThe identification of ever-increasing numbers of RNA species has underlined the importance of robust characterization of bona fide sites of protein-RNA interaction. UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. Here we sought to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, identification of RNA-associated peptides (iRAP), was used to locate 1331 unique RNA-interaction sites at single amino acid residue resolution in 324 S. cerevisiae proteins. Our identified RNA-interaction sites in characterized RNA-protein complex agree well with available high-resolution structures. In known RNA-interacting protiens, 317 sites fall into known and suspected RNA-interaction domains while only 21 sites fall into other annotated sequence features. Strikingly, 993 of the sites identified fall into protein regions that lack any recognizable protein domain structure or annotated sequence features. This suggests that, despite binding RNA in vivo, many of these proteins will not have defined functions in RNA metabolism.\n\nHighlights Method identifies precise RNA-binding sites across proteome\n1331 sites identified that contact RNA in 324 proteins in S. cerevisiae\nRNA binding takes place predominantly outside known protein domains

molecular biology↗

bioRxiv2018

Defining the RNA Interactome by Total RNA-Associated Protein Purification

UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae) and bacteria (E. coli). In all analyses, SILAC labelling was used to quantify protein recovery in the presence and absence of irradiation. For S. cerevisiae, we also compared crosslinking using 254 nm (UVC) irradiation (TRAPP) with 4-thiouracil (4tU) labelling combined with ~350 nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was substantially higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its binding to tRNA loops in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation for highly abundant protein. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified in 395 peptides from 155 unique proteins, following phospho-peptide enrichment combined with a bioinformatics pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.

cell biology↗

bioRxiv2017

HuD is a neural enhancer of global translation acting on mTORC1-responsive genes and sponged by the Y3 small non-coding RNA

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. UV-crosslinking and analysis of cDNA (CRAC) generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells. HuD target sites were identified in 1304 mRNAs, predominantly in the 3UTR, with enrichment for genes involved in protein synthesis and axonogenesis. HuD bound many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlated with the translational efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target was the abundant, small non-coding RNA Y3, which represented 70% of HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its activity. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/205658_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (69K):\norg.highwire.dtl.DTLVardef@17b2d21org.highwire.dtl.DTLVardef@1cfdc8borg.highwire.dtl.DTLVardef@1984f88org.highwire.dtl.DTLVardef@8da1ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗