Biology subjects

Victorino, J. F.

Publications and source records attributed to Victorino, J. F..

3 recordsLinked to original sources

bioRxiv2020

Boosting detection of low abundance proteins in thermal proteome profiling experiments by addition of an isobaric trigger channel to TMT multiplexes

The study of low abundance proteins is a challenge to discovery-based proteomics. Mass-spectrometry (MS) applications, such as thermal proteome profiling (TPP) face specific challenges in detection of the whole proteome as a consequence of the use of nondenaturing extraction buffers. TPP is a powerful method for the study of protein thermal stability, but quantitative accuracy is highly dependent on consistent detection. Therefore, TPP can be limited in its amenability to study low abundance proteins that tend to have stochastic or poor detection by MS. To address this challenge, we incorporated an affinity purified protein complex sample at submolar concentrations as an isobaric trigger channel into a mutant TPP (mTPP) workflow to provide reproducible detection and quantitation of the low abundance subunits of the Cleavage and Polyadenylation Factor (CPF) complex. The inclusion of an isobaric protein complex trigger channel increased detection an average of 40x for previously detected subunits and facilitated detection of CPF subunits that were previously below the limit of detection. Importantly, these gains in CPF detection did not cause large changes in melt temperature (Tm) calculations for other unrelated proteins in the samples, with a high positive correlation between Tm estimates in samples with and without isobaric trigger channel addition. Overall, the incorporation of affinity purified protein complex as an isobaric trigger channel within a TMT multiplex for mTPP experiments is an effective and reproducible way to gather thermal profiling data on proteins that are not readily detected using the original TPP or mTPP protocols.

biochemistry↗

bioRxiv2019

Temperature sensitive Mutant Proteome Profiling: a novel tool for the characterization of the global impact of missense mutations on the proteome

Temperature sensitive (TS) mutants have been foundational in the characterization of essential genes. However, a high-throughput workflow for characterization of biophysical changes in TS mutants is lacking. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of mass spectrometry (MS) based thermal proteome profiling (TPP) to characterize effects of missense mutations on protein stability and PPIs. This study characterizes missense mutations in two different subunits of the 26S proteasome on the thermal stability of the proteome at large, revealing distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. TeMPP is a precise approach to measure changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion.

systems biology↗

bioRxiv2019

RNA Polymerase II CTD phosphatase Rtr1 prevents premature transcription termination

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). Using disruption-compensation (DisCo) protein-protein interaction network analysis, interaction changes were observed within the termination machinery as a consequence of deletion of the serine 5 RNAPII CTD phosphatase Rtr1. Interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1{Delta}, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. These changes could be the result of altered interactions between the termination machinery and/or increased levels of premature termination of RNAPII. Transcriptome analysis in rtr1{Delta} cells found decreased pervasive transcription and a shift in balance of expression of sense and antisense transcripts. Globally, rtr1{Delta} leads to decreases in noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS)-dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes and increased efficiency of snRNA termination. Additionally, premature termination increases globally at protein-coding genes where NNS is recruited during early elongation. The effects of rtr1{Delta} on RNA expression levels were erased following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade terminated noncoding RNAs. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature RNAPII termination of mRNAs and ncRNAs. Additionally, Rtr1 phosphatase activity facilitates low-level elongation of noncoding transcripts that impact the transcriptome through RNAPII interference.\n\nAUTHOR SUMMARYMany cellular RNAs including those that encode for proteins are produced by the enzyme RNA Polymerase II. In this work, we have defined a new role for the phosphatase Rtr1 in the regulation of RNA Polymerase II progression from the start of transcription to the 3 end of the gene where the nascent RNA from protein-coding genes is typically cleaved and polyadenylated. Deletion of the gene that encodes RTR1 leads to changes in the interactions between RNA polymerase II and the termination machinery. Rtr1 loss also causes early termination of RNA Polymerase II at many of its target gene types including protein coding genes and noncoding RNAs. Evidence suggests that the premature termination observed in RTR1 knockout cells occurs through the termination factor and RNA binding protein Nrd1 and its binding partner Nab3. Additionally, many of the prematurely terminated noncoding RNA transcripts are degraded by the Rrp6-containing nuclear exosome, a known component of the Nrd1-Nab3 termination coupled RNA degradation pathway. These findings suggest that Rtr1 normally promotes elongation of RNA Polymerase II transcripts through preventation of Nrd1-directed termination.

systems biology↗