Biology subjects

Petfalski, E.

Publications and source records attributed to Petfalski, E..

4 recordsLinked to original sources

bioRxiv2024

Yeast elongation factor homolog New1 protects a subset of mRNAs from degradation by no-go decay

New1 is a homologue of the essential yeast translation elongation factor eEF3. Lack of New1 has previously been shown to induce queueing of ribosomes upstream of the stop codon on mRNAs encoding specific C-terminal amino acids, primarily lysine and arginine. Here, we used UV crosslinking and analysis of cDNA, long-read nanopore sequencing and proteomics to address the open question of what consequences such queues have for the yeast cell. We show that these queues are ribosomal collisions, which are recognized by the collision sensor and E3 ubiquitin ligase Hel2, marking these collided ribosome complexes for mRNA degradation via canonical no-go decay. Decay is initiated by Cue2-mediated cleavage upstream of the stop codon. Ultimately, this leads to downregulation of encoded proteins, including highly abundant and important metabolic enzymes Pgk1 and Gpm1, as well as translation elongation factors eEF1-alpha and eEF1-beta. Collisions and resulting downstream effects are codon-, rather than amino acid dependent. E.g., for C-terminal lysine and arginine, only specific codons induce collisions upon lack of New1. Our study shows that New1 protects highly abundant and essential genes from degradation by no-go decay thatwould otherwise occur in the absence of translation inhibitors or other direct perturbations of translation.

molecular biology↗

bioRxiv2023

Transcription termination by RNA polymerase I

Transcription elongation is stochastic and driven by a Brownian ratchet mechanism, making it subject to changes in velocity. However, on regions occupied by multiple polymerases, notably the rDNA, DNA rotation plus torsion constrain polymerase molecules to proceed at the same rate generating "torsional entrainment". We report that release of entrainment, by co-transcriptional 3-end cleavage, is permissive for relative movement between polymerases, promoting pausing and backtracking. Subsequent termination (polymerase release) is facilitated by the 5-exonuclease Rat1 (Xrn2) and backtracked transcript cleavage by RNAPI subunit Rpa12. These activities were reproduced in vitro. Short nascent transcripts close to the transcriptional start site, combined with nascent transcript folding energy, similarly facilitate RNAPI pausing. Nascent, backtracked transcripts at pause sites, are targeted by both the exosome cofactor TRAMP and Rat1, promoting termination. Topoisomerase 2 localizes adjacent to RNAPI pause sites, potentially allowing continued elongation by downstream polymerases. Biophysical modeling supported substantial ([~]10%) premature termination. HighlightsNascent pre-rRNA 3 cleavage promotes RNAPI deceleration and termination RNAPI undergoes early, start-site proximal termination at sites of polymerase pausing Biophysical modeling indicates [~]10% early termination - or [~]100 events per minute Model presented for overall organization of pre-rRNA transcription

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

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↗