Amber L. Mosley

Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin

To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression under TOR control.

The SIRT2 Deacetylase Regulates Autoacetylation of p300

Autoacetylation of the p300 histone acetyltransferase controls the transition between VP16-mediated chromatin acetylation and preinitiation complex (PIC) assembly. Currently, it is unknown if and how autoacetylated p300 is deacetylated. We found that the NAD(+)-dependent histone deacetylase SIRT2 deacetylates p300 in vitro and in cells. SIRT2 deacetylates lysine residues in the catalytic domain of p300 and restores binding of p300 to the PIC. RNAi-mediated depletion or chemical inhibition of SIRT2 in cells results in accumulation of acetylated p300. The altered ac-p300/p300 ratio in SIRT2-depleted cells results in decreased p300 recruitment to an integrated VP16-responsive gene and inhibition of transcription. We conclude that p300 undergoes a dynamic cycle of autoacetylation and deacetylation.

Regulation of L-type Ca2+ Channel Activity and Insulin Secretion by the Rem2 GTPase

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions and are controlled by a diversity of intracellular signals. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. In this study, microarray analysis of the mouse insulinoma MIN6 cell line revealed that the transcription of Rem2 gene is strongly induced by exposure to high glucose, which was confirmed by real-time reverse transcriptase-PCR and RNase protection analysis. Because elevation of intracellular Ca2+ in pancreatic β-cells is essential for insulin secretion, we tested the hypothesis that Rem2 attenuates Ca2+ currents to regulate insulin secretion. Co-expression of Rem2 with CaV 1.2 or CaV1.3 L-type Ca + channels in a heterologous expression system completely inhibits de novo Ca2+ current expression. In addition, ectopic overexpression of Rem2 both inhibited L-type Ca2+ channel activity and prevented glucose-stimulated insulin secretion in pancreatic β-cell lines. Co-immunoprecipitation studies demonstrate that Rem2 associates with a variety of CaVβ subunits. Importantly, surface biotinylation studies demonstrate that the membrane distribution of Ca2+ channels was not reduced at a time when channel activity was potently inhibited by Rem2 expression, indicating that Rem2 modulates channel function without interfering with membrane trafficking. Taken together, these data suggest that inhibition of L-type Ca2+ channels by Rem2 signaling may represent a new and potentially important mechanism for regulating Ca2+-triggered exocytosis in hormone-secreting cells, including insulin secretion in pancreatic β-cells.

Repression of transcription by Rgt1 in the absence of glucose requires Std1 and Mth1

Abstract. In the yeast Saccharomyces cerevisiae, glucose induces expression of the hexose transporter (HXT) genes by inhibiting the repressor function of the transcription factor Rgt1. We have previously shown that Rgt1 binds to the HXT gene promoters only in the absence of glucose. In the presence of glucose, Rgt1 becomes phosphorylated and is unable to bind to the HXT promoters and repress their transcription. We report that Rgt1 interacts with Std1 and Mth1 in a yeast two-hybrid assay and co-immunoprecipitates with both proteins in vivo only when glucose is absent. In addition, we demonstrate that repression of HXT gene expression by Rgt1 is abolished in the std1 mth1 double mutant. While Rgt1 is normally phosphorylated only in the presence of high concentrations of glucose, it is constitutively modified in the std1 mth1 double mutant. Based on these data, we conclude that, in the absence of glucose, Rgt1 associates with Std1 and Mth1 to repress HXT gene expression.

Ssu72 Phosphatase-dependent Erasure of Phospho-Ser7 Marks on the RNA Polymerase II C-terminal Domain Is Essential for Viability and Transcription Termination

Background: Reversible phosphorylation of the RNA Polymerase II CTD coordinates co-transcriptional recruitment of factors. Results: Ssu72 is required for erasure of phospho-serine7, and it facilitates Fcp1-mediated phospho-serine2 removal. Conclusion: Removal of phospho-Ser7 mark plays a key role in the transcription cycle. Significance: Persistent negative charge at position 7 of the CTD renders cells non-viable, and Ssu72 plays a prominent role in removing phospho-Ser7. The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) serves an important role in coordinating stage-specific recruitment and release of cellular machines during transcription. Dynamic placement and removal of phosphorylation marks on different residues of a repeating heptapeptide (YSPTSPS) of the CTD underlies the engagement of relevant cellular machinery. Whereas sequential placement of phosphorylation marks is well explored, genome-wide engagement of phosphatases that remove these CTD marks is poorly understood. In particular, identifying the enzyme that erases phospho-Ser7 (Ser7-P) marks is especially important, because we find that substituting this residue with a glutamate, a phospho-mimic, is lethal. Our observations implicate Ssu72 as a Ser7-P phosphatase. We report that removal of all phospho-CTD marks during transcription termination is mechanistically coupled. An inability to remove these marks prevents Pol II from terminating efficiently and will likely impede subsequent assembly into the pre-initiation complex.

A label free quantitative proteomic analysis of the Saccharomyces cerevisiae nucleus

To gain insight into the nuclear proteome of Saccharomyces cerevisiae, nuclei were isolated and fractionated via sucrose gradient sedimentation. The resulting fractions were analyzed using multidimensional protein identification technology and the detected proteins were quantified using normalized spectral counts. A large number of low abundance proteins, many of which are involved in transcriptional regulation, were recovered. Sucrose gradient elution profiles of known protein complex components demonstrated that this approach may provide insight into the question of what percentage of the total population of a protein is in one complex, versus another protein complex, or exists as a free protein.

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.

The Exosome Component Rrp6 Is Required for RNA Polymerase II Termination at Specific Targets of the Nrd1-Nab3 Pathway

The exosome and its nuclear specific subunit Rrp6 form a 3’-5’ exonuclease complex that regulates diverse aspects of RNA biology including 3’ end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3’ end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3’ end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3’ RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3’ end RNA processing and/or degradation at specific targets.

RPRD1A and RPRD1B are human RNA polymerase II C-terminal domain scaffolds for Ser5 dephosphorylation

The RNA polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeats (1-YSPTSPS-7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD-interaction domains (CIDs) with RNAPII CTD repeats phosphorylated at S2 and S7. Crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with RNAPII CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.

Rtr1 Is a Dual Specificity Phosphatase That Dephosphorylates Tyr1 and Ser5 on the RNA Polymerase II CTD

The phosphorylation state of heptapeptide repeats within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (PolII) controls the transcription cycle and is maintained by the competing action of kinases and phosphatases. Rtr1 was recently proposed to be the enzyme responsible for the transition of PolII into the elongation and termination phases of transcription by removing the phosphate marker on serine 5, but this attribution was questioned by the apparent lack of enzymatic activity. Here we demonstrate that Rtr1 is a phosphatase of new structure that is auto-inhibited by its own C-terminus. The enzymatic activity of the protein in vitro is functionally important in vivo as well: a single amino acid mutation that reduces activity leads to the same phenotype in vivo as deletion of the protein-coding gene from yeast. Surprisingly, Rtr1 dephosphorylates not only serine 5 on the CTD but also the newly described anti-termination tyrosine 1 marker, supporting the hypothesis that Rtr1 and its homologs promote the transition from transcription to termination.