CDK12 Activity-Dependent Phosphorylation Events in Human Cells.

We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition.

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation.

Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

Human CDK12 and CDK13, multi-tasking CTD kinases for the new millenium.

As the new millennium began, CDK12 and CDK13 were discovered as nucleotide sequences that encode protein kinases related to cell cycle CDKs. By the end of the first decade both proteins had been qualified as CTD kinases, and it was emerging that both are heterodimers containing a Cyclin K subunit. Since then, many studies on CDK12 have shown that, through phosphorylating the CTD of transcribing RNAPII, it plays critical roles in several stages of gene expression, notably RNA processing; it is also crucial for maintaining genome stability. Fewer studies on CKD13 have clearly shown that it is functionally distinct from CDK12. CDK13 is important for proper expression of a number of genes, but it also probably plays yet-to-be-discovered roles in other processes. This review summarizes much of the work on CDK12 and CDK13 and attempts to evaluate the results and place them in context. Our understanding of these two enzymes has begun to mature, but we still have much to learn about both. An indicator of one major area of medically-relevant future research comes from the discovery that CDK12 is a tumor suppressor, notably for certain ovarian and prostate cancers. A challenge for the future is to understand CDK12 and CDK13 well enough to explain how their loss promotes cancer development and how we can intercede to prevent or treat those cancers. Abbreviations: CDK: cyclin-dependent kinase; CTD: C-terminal repeat domain of POLR2A; CTDK-I: CTD kinase I (yeast); Ctk1: catalytic subunit of CTDK-I; Ctk2: cyclin-like subunit of CTDK-I; PCAP: phosphoCTD-associating protein; POLR2A: largest subunit of RNAPII; SRI domain: Set2-RNAPII Interacting domain.

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas.

The RNA Polymerase II C-terminal domain (CTD) kinase CDK12 has been implicated as a tumor suppressor and regulator of DNA damage response genes. Although much has been learned about CDK12 and its activity, due to the lack of a specific inhibitor and the complications posed by long term RNAi depletion, much is still unknown about the particulars of CDK12 function. Therefore gaining a better understanding of CDK12's roles at the molecular level will be challenging without the development of additional tools. In order to address these issues we have used the CRISPR/Cas gene engineering system to create a mammalian cell line in which the only functional copy of CDK12 is selectively inhibitable by a cell-permeable adenine analog (analog-sensitive CDK12). Inhibition of CDK12 results in a perturbation of the phosphorylation patterns on the CTD and an arrest in cellular proliferation. This cell line should serve as a powerful tool for future studies.

Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex.

The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3'-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a "2A" peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.

A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II.

RNA polymerase II translocates across much of the genome and since it can be blocked by many kinds of DNA lesions, detects DNA damage proficiently; it thereby contributes to DNA repair and to normal levels of DNA damage resistance. However, the components and mechanisms that respond to polymerase blockage are largely unknown, except in the case of UV-induced damage that is corrected by nucleotide excision repair. Because elongating RNAPII carries with it numerous proteins that bind to its hyperphosphorylated CTD, we tested for effects of interfering with this binding. We find that expressing a decoy CTD-carrying protein in the nucleus, but not in the cytoplasm, leads to reduced DNA damage resistance. Likewise, inducing aberrant phosphorylation of the CTD, by deleting CTK1, reduces damage resistance and also alters rates of homologous recombination-mediated repair. In line with these results, extant data sets reveal a remarkable, highly significant overlap between phosphoCTD-associating protein genes and DNA damage-resistance genes. For one well-known phosphoCTD-associating protein, the histone methyltransferase Set2, we demonstrate a role in DNA damage resistance, and we show that this role requires the phosphoCTD binding ability of Set2; surprisingly, Set2's role in damage resistance does not depend on its catalytic activity. To explain all of these observations, we posit the existence of a CTD-Associated DNA damage Response (CAR) system, organized around the phosphoCTD of elongating RNAPII and comprising a subset of phosphoCTD-associating proteins.

Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat.

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N-terminal WW domains and the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II through its C-terminal FF domains. Here, we report biochemical and structural characterization of the C-terminal three FF domains (FF4-6) of TCERG1, revealing a rigid integral domain structure of the tandem FF repeat that interacts with the hyperphosphorylated CTD (PCTD). Although FF4 and FF5 adopt a classical FF domain fold containing three orthogonally packed α helices and a 310 helix, FF6 contains an additional insertion helix between α1 and α2. The formation of the integral tandem FF4-6 repeat is achieved by merging the last helix of the preceding FF domain and the first helix of the following FF domain and by direct interactions between neighboring FF domains. Using peptide column binding assays and NMR titrations, we show that binding of the FF4-6 tandem repeat to the PCTD requires simultaneous phosphorylation at Ser(2), Ser(5), and Ser(7) positions within two consecutive Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events.

Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease.

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.

Phosphorylation of RNAPII: To P-TEFb or not to P-TEFb?

The C-terminal domain of RNA polymerase II undergoes a cycle of phosphorylation which allows it to temporally couple transcription with transcription-associated processes. The characterization of hitherto unrecognized metazoan elongation phase CTD kinase activities expands our understanding of this coupling. We discuss the circumstances that delayed the recognition of these kinase activities.

Cotranscriptional association of mRNA export factor Yra1 with C-terminal domain of RNA polymerase II.

The unique C-terminal domain (CTD) of RNA polymerase II, composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the phosphorylated CTD and use it to localize to nascent pre-mRNA during transcription. In Saccharomyces cerevisiae, the mRNA export protein Yra1 (ALY/RNA export factor in metazoa) cotranscriptionally associates with mRNA and delivers it to the nuclear pore complex for export to the cytoplasm. Here we report that Yra1 directly binds in vitro the hyperphosphorylated form of the CTD characteristic of elongating RNA polymerase II and contains a phospho-CTD-interacting domain within amino acids 18-184, which also include an "RNA recognition motif" (RRM) (residues 77-184). Using UV cross-linking, we showed that the RRM alone binds RNA, although a larger segment extending to the C terminus (amino acids 77-226) displayed stronger RNA binding activity. Although the RRM is implicated in both RNA and CTD binding, RRM point mutations separated these two functions. Both functions are important in vivo as RNA binding-defective or CTD binding-defective versions of Yra1 engendered growth and mRNA export defects. We also report the construction and characterization of a useful new temperature-sensitive YRA1 allele (R107A/F126A). Using ChIP, we demonstrated that removing the N-terminal 76 amino acids of Yra1 (all of the phospho-CTD-interacting domain up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the phospho-CTD is likely involved directly in the cotranscriptional recruitment of Yra1.

Updating the CTD Story: From Tail to Epic.

Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA but also coordinates transcription-related processes via its unique C-terminal repeat domain (CTD). The CTD is an RNAPII-specific protein segment consisting of repeating heptads with the consensus sequence Y(1)S(2)P(3)T(4)S(5)P(6)S(7) that has been shown to be extensively post-transcriptionally modified in a coordinated, but complicated, manner. Recent discoveries of new modifications, kinases, and binding proteins have challenged previously established paradigms. In this paper, we examine results and implications of recent studies related to modifications of the CTD and the respective enzymes; we also survey characterizations of new CTD-binding proteins and their associated processes and new information regarding known CTD-binding proteins. Finally, we bring into focus new results that identify two additional CTD-associated processes: nucleocytoplasmic transport of mRNA and DNA damage and repair.

cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72.

RNA polymerase II coordinates co-transcriptional events by recruiting distinct sets of nuclear factors to specific stages of transcription via changes of phosphorylation patterns along its C-terminal domain (CTD). Although it has become increasingly clear that proline isomerization also helps regulate CTD-associated processes, the molecular basis of its role is unknown. Here, we report the structure of the Ser(P)(5) CTD phosphatase Ssu72 in complex with substrate, revealing a remarkable CTD conformation with the Ser(P)(5)-Pro(6) motif in the cis configuration. We show that the cis-Ser(P)(5)-Pro(6) isomer is the minor population in solution and that Ess1-catalyzed cis-trans-proline isomerization facilitates rapid dephosphorylation by Ssu72, providing an explanation for recently discovered in vivo connections between these enzymes and a revised model for CTD-mediated small nuclear RNA termination. This work presents the first structural evidence of a cis-proline-specific enzyme and an unexpected mechanism of isomer-based regulation of phosphorylation, with broad implications for CTD biology.

CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1.

Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 3' end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 ↔ CDK9 and Ctk1 ↔ CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.

RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription.

It is known that transcription can induce DNA recombination, thus compromising genomic stability. RECQ5 DNA helicase promotes genomic stability by regulating homologous recombination. Recent studies have shown that RECQ5 forms a stable complex with RNA polymerase II (RNAPII) in human cells, but the cellular role of this association is not understood. Here, we provide evidence that RECQ5 specifically binds to the Ser2,5-phosphorylated C-terminal repeat domain (CTD) of the largest subunit of RNAPII, RPB1, by means of a Set2-Rpb1-interacting (SRI) motif located at the C-terminus of RECQ5. We also show that RECQ5 associates with RNAPII-transcribed genes in a manner dependent on the SRI motif. Notably, RECQ5 density on transcribed genes correlates with the density of Ser2-CTD phosphorylation, which is associated with the productive elongation phase of transcription. Furthermore, we show that RECQ5 negatively affects cell viability upon inhibition of spliceosome assembly, which can lead to the formation of mutagenic R-loop structures. These data indicate that RECQ5 binds to the elongating RNAPII complex and support the idea that RECQ5 plays a role in the maintenance of genomic stability during transcription.

Genetic organization, length conservation, and evolution of RNA polymerase II carboxyl-terminal domain.

With a simple tandem iterated sequence, the carboxyl terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II) serves as the central coordinator of mRNA synthesis by harmonizing a diversity of sequential interactions with transcription and processing factors. Despite intense research interest, many key questions regarding functional and evolutionary constraints on the CTD remain unanswered; for example, what selects for the canonical heptad sequence, its tandem array across organismal diversity, and constant CTD length within given species and finally and how a sequence-identical, repetitive structure can orchestrate a diversity of simultaneous and sequential, stage-dependent interactions with both modifying enzymes and binding partners? Here we examine comparative sequence evolution of 58 RNAP II CTDs from diverse taxa representing all six major eukaryotic supergroups and employ integrated evolutionary genetic, biochemical, and biophysical analyses of the yeast CTD to further clarify how this repetitive sequence must be organized for optimal RNAP II function. We find that the CTD is composed of indivisible and independent functional units that span diheptapeptides and not only a flexible conformation around each unit but also an elastic overall structure is required. More remarkably, optimal CTD function always is achieved at approximately wild-type CTD length rather than number of functional units, regardless of the characteristics of the sequence present. Our combined observations lead us to advance an updated CTD working model, in which functional, and therefore, evolutionary constraints require a flexible CTD conformation determined by the CTD sequence and tandem register to accommodate the diversity of CTD-protein interactions and a specific CTD length rather than number of functional units to correctly order and organize global CTD-protein interactions. Patterns of conservation of these features across evolutionary diversity have important implications for comparative RNAP II function in eukaryotes and can more clearly direct specific research on CTD function in currently understudied organisms.

The phosphoCTD-interacting domain of Topoisomerase I.

The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.

Comparative genome-wide screening identifies a conserved doxorubicin repair network that is diploid specific in Saccharomyces cerevisiae.

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Delta, ctf4Delta, ctk1Delta, hfi1Delta,nup133Delta, tho2Delta) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Delta and hfi1Delta) or deficient (tho2Delta) for recombination. Using these and other "THO2-like" hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Delta, thp1Delta, thp2Delta) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases.

Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.

The essential sequence elements required for RNAP II carboxyl-terminal domain function in yeast and their evolutionary conservation.

The carboxyl-terminal domain (CTD) of eukaryotic RNA polymerase II is the staging platform for numerous proteins involved in transcription initiation, mRNA processing, and general coordination of nuclear events. Concordant with these central roles in cellular metabolism, the consensus sequence, tandemly repeated structure, and core functions of the CTD are conserved across diverse eukaryotic lineages; however, in other eukaryotes, the CTD has been allowed to degenerate completely. Even in groups where the CTD is strongly conserved, genetic analyses and comparative genomic investigations show that a variety of individual substitutions and insertions are permissible. Therefore, the specific functional constraints reflected by the CTD's conservation across much of eukaryotic evolution have remained somewhat puzzling. Here we propose a hypothesis to explain that strong conservation in budding yeast, based on both comparative and experimental evidence. Through genetic complementation for CTD function, we identify 2 sequence elements contained within pairs of heptapeptides, "Y(1)-Y(8)" and "S(2)-S(5)-S(9)," which are required for all essential CTD functions in yeast. The dual requirements of these motifs can account for strong purifying selection on the canonical CTD heptapeptide. Further, in vitro analysis of GST-CTD fusion proteins as substrates for multiple CTD-directed kinases show reduced phosphorylation efficiencies with increased distance between functional units. This indicates that requirements of the RNAP II phosphorylation cycle are most likely responsible for the strong purifying selection on tandemly repeated CTD structure.