Olga Calvo

Sub1 associates with Spt5 and influences RNA polymerase II transcription elongation rate

The transcriptional coactivator Sub1 is a functional component of the PIC implicated in several steps of mRNA metabolism. Sub1 directly and specifically influences the transcription elongation rate by an interaction with the elongation factor Spt5. The results provide a novel mechanistic insight into the process of elongation by RNAPII.

Heterogeneous decrease of bone mineral density in the vertebral column of ovariectomized rats.

The long-term effect of ovariectomy on the loss of bone mineral density (BMD) was evaluated in rats with and without estrogen treatment; BMD was studied in the lumbar and caudal vertebrae, measured by DXA, to find how the losses of BMD occur in the axial skeleton. Seventy female Wistar rats of 3 months of age were divided into four groups as follows: group 1: control animals; group 2: ovariectomized animals; group 3: ovariectomized animals undergoing treatment with estrogen (0.25 mg/kg per week of 17-beta estradiol); group 4: ovariectomized rats undergoing estrogen treatment only during the last 3 months of the experimental period. No significant differences were found among the groups in regard to the BMD values of the caudal vertebrae at either 3 or 6 months. Likewise, in the lumbar vertebrae there were no significant differences among the groups after 3 months. However, at 6 months, a decrease in the BMDs of the ovariectomized animals with respect to the remaining groups was found: 226 +/- 11 mg/cm2 in the ovariectomized group; 262 +/- 14 mg/cm2 in the controls; 255 +/- 4 mg/cm2 in the rats receiving estrogen treatment for 6 months; and 259 +/- 5 mg/cm2 in the animals receiving estrogen for 3 months. The study also reveals the absence of differences in the bone mineral density between the ovariectomized and control rats when the former received estrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association. DOI: http://dx.doi.org/10.7554/eLife.20832.001

Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. However, their mechanism(s) of action remains unclear. Here, we show that the Rpb4/7 heterodimer in Saccharomyces cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Proper phosphorylation of the CTD is critical for the synthesis and processing of RNAPII transcripts. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7 and Thr4 within the CTD. RPB4 interacted genetically with genes encoding CTD phosphatases (SSU72, FCP1), CTD kinases (KIN28, CTK1, SRB10) and a prolyl isomerase that targets the CTD (ESS1). We show that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Our data also suggest that Fcp1 is the Thr4P phosphatase in yeast. Based on these and other results, we suggest a model in which Rpb4/7 helps recruit and potentially stimulate the activity of CTD-modifying enzymes, a role that is central to RNAPII function.

The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation

RNA polymerase (pol) II establishes many protein–protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the “foot” domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme.

Sub1 contacts the RNA polymerase II stalk to modulate mRNA synthesis.

Abstract Biogenesis of messenger RNA is critically influenced by the phosphorylation state of the carboxy-terminal domain (CTD) in the largest RNA polymerase II (RNAPII) subunit. Several kinases and phosphatases are required to maintain proper CTD phosphorylation levels and, additionally, several other proteins modulate them, including Rpb4/7 and Sub1. The Rpb4/7 heterodimer, constituting the RNAPII stalk, promote phosphatase functions and Sub1 globally influences CTD phosphorylation, though its mechanism remains mostly unknown. Here, we show that Sub1 physically interacts with the RNAPII stalk domain, Rpb4/7, likely through its C-terminal region, and associates with Fcp1. While Rpb4 is not required for Sub1 interaction with RNAPII complex, a fully functional heterodimer is required for Sub1 association to promoters. We also demonstrate that a complete CTD is necessary for proper association of Sub1 to chromatin and to the RNAPII. Finally, genetic data show a functional relationship between Sub1 and the RNAPII clamp domain. Altogether, our results indicate that Sub1, Rpb4/7 and Fcp1 interaction modulates CTD phosphorylation. In addition, Sub1 interaction with Rpb4/7 can also modulate transcription start site selection and transcription elongation rate likely by influencing the clamp function.

Sub1/PC4, a multifaceted factor: from transcription to genome stability

Yeast Sub1 and human PC4, two DNA-binding proteins, were originally identified as transcriptional coactivators with a role during transcription preinitiation/initiation. Indeed, Sub1 is a PIC component, and both PC4 and Sub1 also influence the initiation–elongation transition. Moreover, in the specific case of Sub1, it has been clearly reported that it influences processes downstream during mRNA biogenesis, such as transcription elongation, splicing and termination, and even RNAPII phosphorylation/dephosphorylation. Although Sub1 mechanism of action has been mostly unknown up to date, thanks to the recent finding that Sub1 directly interacts with the RNAPII stalk domain, we can envision how it can modulate so many processes. In addition, Sub1 and PC4 participate in RNAPIII transcription as well, and much additional evidence indicates an evolutionarily conserved role for Sub1 and PC4 in the maintenance of genome stability. In this regard, the most novel function of Sub1 and PC4 has been related to the ability of these proteins to bind G-quadruplex DNA structures that may arise as a consequence of the transcription process.

Genetic analysis of RNA polymerase I unveils new role of the Rpa12 subunit during transcription

Most transcriptional activity of exponentially growing cells is carried out by RNA Polymerase I (Pol I), which produces a large rRNA precursor. The Pol I transcription cycle is achieved through complex structural rearrangements of the enzyme, revealed by recent structural studies. In the yeast S. cerevisiae the Pol 1 subunit Rpa49, particularly its C-terminal tandem winged helix domain (Rpa49Ct), is required supports both initiation and elongation of the transcription cycle. Here, we characterized novel extragenic suppressors of the growth defect caused by the absence of Rpa49. We identified suppressor mutations on the two largest subunits of Pol I, Rpa190 and Rpa135, as well as Rpa12. Suppressor mutants RPA135-F301S and RPA12-S6L restored normal rRNA synthesis and increased Pol I density on rDNA genes in the absence of Rpa49Ct. Most mutated residues cluster at an interface formed by the jaw in Rpa190, the lobe in Rpa135, and subunit Rpa12 when mapped on the structure of Pol I. Our genetic data in S. cerevisiae suggest a new role for Rpa12 at the jaw/lobe interface during transcription cycle.Abstract Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I. Author summary The nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

Structural basis of RNA polymerase I stalling at UV light-induced DNA damage

Significance DNA lesions threaten cellular life and must be repaired to maintain genome integrity. During transcription, RNA polymerases (RNAPs) actively scan DNA to find bulky lesions and trigger their repair. In growing eukaryotic cells, most transcription involves synthesis of ribosomal RNA by RNAP I (Pol I), and Pol I activity thus influences survival upon DNA damage. We determined the high-resolution electron cryomicroscopy structure of Pol I stalled by a UV-induced lesion, cyclobutane pyrimidine dimer (CPD), to unveil how the enzyme manages this important DNA damage. We found that Pol I gets stalled when the lesion reaches the bridge helix, a structural element involved in enzyme advance along DNA. We identified Pol I-specific residues around the active site that contribute to CPD-induced arrest. RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition

Abstract Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/β domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination.