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.

Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation.

Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.

Quantitative proteomics demonstrates that the RNA polymerase II subunits Rpb4 and Rpb7 dissociate during transcriptional elongation.

Eukaryotic RNA polymerase II (RNAPII) is a 12-subunit enzyme that is responsible for the transcription of messenger RNA. Two of the subunits of RNA polymerase II, Rpb4 and Rpb7, have been shown to dissociate from the enzyme under a number of specific laboratory conditions. However, a biological context for the dissociation of Rpb4 and Rpb7 has not been identified. We have found that Rpb4/7 dissociate from RNAPII upon interaction with specific transcriptional elongation-associated proteins that are recruited to the hyperphosphorylated form of the C-terminal domain. However, the dissociation of Rpb4/7 is likely short lived because a significant level of free Rpb4/7 was not detected by quantitative proteomic analyses. In addition, we have found that RNAPII that is isolated through Rpb7 is depleted in serine 2 C-terminal domain phosphorylation. In contrast to previous reports, these data indicate that Rpb4/7 are dispensable during specific stages of transcriptional elongation in Saccharomyces cerevisiae.

Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells.

We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.

The gas-phase ligand exchange reactions of cobalt and zinc acetylacetonate, hexafluoroacetylacetonate, and trifluorotrimethylacetylacetonate complexes.

The gas-phase ligand exchange reactions between Co(II) and Zn(II) complexes containing the acetylacetonate (acac), hexafluoroacetylacetonate (hfac), and trifluorotrimethylacetylacetonate (tftm) ligands were investigated using a triple quadrupole mass spectrometer. The gas-phase mixed ligand products of [Cu(acac)(tftm)](+), [Ni(acac)(tftm)](+), [Cu(hfac)(tftm)](+), and [Ni(hfac)(tftm)](+) were formed following the co-sublimation of either homo-metal or hetero-metal precursors and are reported herein for the first time. The fragmentation patterns of these mixed ligand species along with those of Cu(tftm)(2) and Ni(tftm)(2) are also presented. The collision cell of the instrument was utilized to examine the gas-phase reactions between mass-selected ions and specific neutral target compounds.

The gas-phase ligand exchange of copper and nickel acetylacetonate, hexafluoroacetylacetonate and trifluorotrimethylacetylacetonate complexes.

The gas-phase ligand-exchange reactions between Cu(II) and Ni(II) complexes containing the acetylacetonate (acac), hexafluoroacetylacetonate (hfac), and trifluorotrimethylacetylacetonate (tftm) ligands were investigated using a triple quadrupole mass spectrometer. The gas-phase mixed-ligand products of [Cu(acac)(tftm)](+), [Ni(acac)(tftm)](+), [Cu(hfac)(tftm)](+), and [Ni(hfac)(tftm)](+) were formed following the co-sublimation of either homo-metal or hetero-metal precursors. The gas-phase formation of [Cu(acac)(tftm)](+), [Cu(hfac)(tftm)](+), [Ni(acac)(tftm)](+), and [Ni(hfac)(tftm)](+) complexes is reported herein for the first time. The corresponding fragmentation patterns of these species along with those of Cu(tftm)(2) and Ni(tftm)(2) are also presented. Mass-selected ion-neutral reactions were investigated.

Bis[µ-pentane-2,4-dionato(1-)]bis-{aqua-[1,1,1,5,5,5-hexa-fluoro-pentane-2,4-dionato(1-)]cobalt(II)}.

The title complex, [Co(2)(C(5)HF(6)O(2))(2)(C(5)H(7)O(2))(2)(H(2)O)(2)], is centrosymmetric with a crystallographic inversion center in the middle of the mol-ecule. The octa-hedrally coordinated Co(II) atoms are bridged by two chelating acetyl-acetonate (acac) ligands and two more electron-poor 1,1,1,5,5,5-hexa-fluoro-pentane-2,4-dionato (hfac) ligands are bonded terminally in a solely chelating manner. The coordinated water mol-ecules form inter-molecular O-H⋯O hydrogen bonds with electron-rich acac O atoms of neighboring mol-ecules, leading to strings of mol-ecules along the a axis.

cis-[Aqua/methanol(0.45/1.55)](1,1,1-trifluoro-5,5-dimethyl-hexane-2,4-dionato)nickel(II)-cis-[aqua/methanol(1.49/0.51)](1,1,1-trifluoro-5,5-dimethyl-hexane-2,4-dionato)nickel(II) (1/1).

The title compound, [Ni(C(8)H(10)F(3)O(2))(2)(CH(4)O)(1.55)(H(2)O)(0.45)][Ni(C(8)H(10)F(3)O(2))(2)(CH(4)O)(0.51)(H(2)O)(1.49)], is an octa-hedral nickel(II) complex with two acetyl-acetonate-like 1,1,1-trifluoro-5,5-dimethyl-hexane-2,4-dionate ligands. The two chelating ligands are in cis positions with respect to each other and the remaining two adjacent coordination sites are taken up by water and methanol donor mol-ecules. In both crystallographically independent mol-ecules, each donor site shows disorder of methanol and water with occupancies of 0.51 (1) and 0.55 (1) in favor of methanol. The remaining two donor sites are not disordered and are water for the first and methanol for the second independent mol-ecule. Rotational disorder is observed for one of the tert-butyl groups, the occupancy rate for the major component here is 0.687 (9). O-H⋯O hydrogen bonds connect the two independent mol-ecules with each other and, across a crystallographic inversion center, they are combined with two neighboring mol-ecules to form a centrosymmetric hydrogen-bonded tetra-mer.

trans-Dimethano-lbis(1,1,1-trifluoro-5,5-dimethyl-hexane-2,4-dionato)zinc(II).

The title compound, [Zn(C(8)H(10)F(3)O(2))(2)(CH(4)O)(2)], is a dimethanol coordinated zinc complex with the acetyl acetonate derivative 1,1,1-trifluoro-5,5-dimethyl-hexane-2,4-dionate. The bis--ß-diketonate complex, which is isostructural with its Co analogue, is located on a crystallographic inversion center. The complex is octa-hedral with basically no distortion, and the methanol mol-ecules are in trans positions with respect to one another. The planes of the ß-diketonate and the ZnO(4) unit are tilted by 18.64 (10)° against each other. O-H⋯O hydrogen bonds between the methanol hydroxyl groups and neighboring diketonate O atoms create chains running along [100].

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes.

The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae.

The phosphatase Rtr1 has been implicated in dephosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) during transcription elongation and in regulation of nuclear import of RNAPII. Although it has been shown that Rtr1 interacts with RNAPII in yeast and humans, the specific mechanisms that underlie Rtr1 recruitment to RNAPII have not been elucidated. To address this, we have performed an in-depth proteomic analysis of Rtr1 interacting proteins in yeast. Our studies revealed that hyperphosphorylated RNAPII is the primary interacting partner for Rtr1. To extend these findings, we performed quantitative proteomic analyses of Rtr1 interactions in yeast strains deleted for CTK1, the gene encoding the catalytic subunit of the CTD kinase I (CTDK-I) complex. Interestingly, we found that the interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains. We hypothesize that serine-2 CTD phosphorylation is required for Rtr1 recruitment to RNAPII during transcription elongation.

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.