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 the 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 40× 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 an affinity-purified protein complex as an isobaric trigger channel within a tandem mass tag (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.

Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome.

Temperature-sensitive (TS) missense mutants have been foundational for characterization of essential gene function. However, an unbiased approach for analysis of biochemical and biophysical changes in TS missense mutants within the context of their functional proteomes is lacking. We applied MS-based thermal proteome profiling (TPP) to investigate the proteome-wide effects of missense mutations in an application that we refer to as mutant thermal proteome profiling (mTPP). This study characterized global impacts of temperature sensitivity-inducing missense mutations in two different subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and global proteomics were similar between the mutants, which could suggest that a similar functional disruption is occurring in both missense variants. Results from mTPP, however, provide unique insights into the mechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were not obtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP 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. Although experiments were performed under permissive conditions, mTPP provided insights into the underlying protein stability changes that cause dramatic cellular phenotypes observed at nonpermissive temperatures. Overall, mTPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, nonbiased fashion.

RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination.

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous 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. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome.

Writing a wrong: Coupled RNA polymerase II transcription and RNA quality control.

Processing and maturation of precursor RNA species is coupled to RNA polymerase II transcription. Co-transcriptional RNA processing helps to ensure efficient and proper capping, splicing, and 3' end processing of different RNA species to help ensure quality control of the transcriptome. Many improperly processed transcripts are not exported from the nucleus, are restricted to the site of transcription, and are in some cases degraded, which helps to limit any possibility of aberrant RNA causing harm to cellular health. These critical quality control pathways are regulated by the highly dynamic protein-protein interaction network at the site of transcription. Recent work has further revealed the extent to which the processes of transcription and RNA processing and quality control are integrated, and how critically their coupling relies upon the dynamic protein interactions that take place co-transcriptionally. This review focuses specifically on the intricate balance between 3' end processing and RNA decay during transcription termination. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Processing > 3' End Processing RNA Processing > Splicing Mechanisms RNA Processing > Capping and 5' End Modifications.