RNA polymerase II speed: a key player in controlling and adapting transcriptome composition.

RNA polymerase II (RNA Pol II) speed or elongation rate, i.e., the number of nucleotides synthesized per unit of time, is a major determinant of transcriptome composition. It controls co-transcriptional processes such as splicing, polyadenylation, and transcription termination, thus regulating the production of alternative splice variants, circular RNAs, alternatively polyadenylated transcripts, or read-through transcripts. RNA Pol II speed itself is regulated in response to intra- and extra-cellular stimuli and can in turn affect the transcriptome composition in response to these stimuli. Evidence points to a potentially important role of transcriptome composition modification through RNA Pol II speed regulation for adaptation of cells to a changing environment, thus pointing to a function of RNA Pol II speed regulation in cellular physiology. Analyzing RNA Pol II speed dynamics may therefore be central to fully understand the regulation of physiological processes, such as the development of multicellular organisms. Recent findings also raise the possibility that RNA Pol II speed deregulation can be detrimental and participate in disease progression. Here, we review initial and current approaches to measure RNA Pol II speed, as well as providing an overview of the factors controlling speed and the co-transcriptional processes which are affected. Finally, we discuss the role of RNA Pol II speed regulation in cell physiology.

3' end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells.

3' end formation of pre-mRNAs is coupled to their transcription via the C-terminal domain (CTD) of RNA polymerase II (Pol II). Nearly all protein-coding transcripts are matured by cleavage and polyadenylation (CPA), which is frequently misregulated in disease. Understanding how transcription is coordinated with CPA in human cells is therefore very important. We found that the CTD is heavily phosphorylated on Ser2 (Ser2p) at poly(A) (pA) signals coincident with recruitment of the CstF77 CPA factor. Depletion of the Ser2 kinase Cdk12 impairs Ser2p, CstF77 recruitment, and CPA, strongly suggesting that the processes are linked, as they are in budding yeast. Importantly, we additionally show that the high Ser2p signals at the 3' end depend on pA signal function. Down-regulation of CPA results in the loss of a 3' Ser2p peak, whereas a new peak is formed when CPA is induced de novo. Finally, high Ser2p signals are generated by Pol II pausing, which is a well-known feature of pA site recognition. Thus, a reciprocal relationship between early steps in pA site processing and Ser2p ensures efficient 3' end formation.

Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence.

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence.

RNA elements directing in vivo assembly of the 7SK/MePCE/Larp7 transcriptional regulatory snRNP.

Through controlling the nuclear level of active positive transcription elongation factor b (P-TEFb), the 7SK small nuclear RNA (snRNA) functions as a key regulator of RNA polymerase II transcription. Together with hexamethylene bisacetamide-inducible proteins 1/2 (HEXIM1/2), the 7SK snRNA sequesters P-TEFb into transcriptionally inactive ribonucleoprotein (RNP). In response to transcriptional stimulation, the 7SK/HEXIM/P-TEFb RNP releases P-TEFb to promote polymerase II-mediated messenger RNA synthesis. Besides transiently associating with HEXIM1/2 and P-TEFb, the 7SK snRNA stably interacts with the La-related protein 7 (Larp7) and the methylphosphate capping enzyme (MePCE). In this study, we used in vivo RNA-protein interaction assays to determine the sequence and structural elements of human 7SK snRNA directing assembly of the 7SK/MePCE/Larp7 core snRNP. MePCE interacts with the short 5'-terminal G1-U4/U106-G111 helix-tail motif and Larp7 binds to the 3'-terminal hairpin and the following U-rich tail of 7SK. The overall RNA structure and some particular nucleotides provide the information for specific binding of MePCE and Larp7. We also demonstrate that binding of Larp7 to 7SK is a prerequisite for in vivo recruitment of P-TEFb, indicating that besides providing stability for 7SK, Larp7 directly participates in P-TEFb regulation. Our results provide further explanation for the frequently observed link between Larp7 mutations and cancer development.

Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat.

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

[Misregulation of P-TEFb activity: pathological consequences]. / Perturbations de la transcription liées à une dérégulation de P-TEFb : cancer, Sida et hypertrophie cardiaque.

P-TEFb stimulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA pol II and antagonizing the effects of negative elongation factors. Its cellular availability is controlled by an abundant non coding RNA, conserved through evolution, the 7SK RNA. Together with the HEXIM proteins, 7SK RNA associates with and sequesters a fraction of cellular P-TEFb into a catalytically inactive complex. Active and inactive forms of P-TEFb are kept in a functional and dynamic equilibrium tightly linked to the transcriptional requirement of the cell. Importantly, cardiac hypertrophy and development of various types of human malignancies have been associated with increased P-TEFb activity, consequence of a disruption of this regulatory equilibrium. In addition, the HIV-1 Tat protein also releases P-TEFb from the 7SK/HEXIM complex during viral infection to promote viral transcription and replication. Here, we review the roles played by the 7SK RNP in cancer development, cardiac hypertrophy and AIDS.

Small Interfering RNAs Targeting a Chromatin-Associated RNA Induce Its Transcriptional Silencing in Human Cells.

Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, including plants and yeast. In mammals, its extent remains somewhat debated. Previous studies showed that siRNAs targeting gene promoters could induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by using nascent RNA capture and RNA polymerase II chromatin immunoprecipitation, we show that siRNAs targeting a chromatin-associated noncoding RNA induced its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further showed that this silencing was due to base-pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we showed that only the wild-type allele, but not the deleted allele, was silenced by the siRNA, indicating that transcriptional silencing occurred only in cis. Finally, we demonstrated that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at a distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.

Molecular characterization and expression analysis of the Rab GTPase family in Vitis vinifera reveal the specific expression of a VvRabA protein.

As a first step to investigate whether Rab GTPases are involved in grape berry development, the Vitis vinifera EST and gene databases were searched for members of the VvRab family. The grapevine genome was found to contain 26 VvRabs that could be distributed into all of the eight groups described in the literature for model plants. Genetic mapping was successfully performed; VvRabs were mostly located on independent chromosomes, apart from eight that were located on the as yet unassigned portions of the genome clustered in the ChrUn Random chromosome. Conserved and divergent regions between VvRab protein sequences were identified. Transcript expression of 11 VvRabs was analysed by real-time quantitative RT-PCR. Except for VvRabA5b, transcript expression was detected, in general, in all the organs investigated, but with different patterns. In grape berries, VvRab transcripts were expressed at all stages of fruit development, with different profiles, except in the case of members of the A family which displayed generally similar patterns. The response to growth regulators in cell cultures was generally specific to each VvRab, with a differential pattern of expression for ethylene, auxin, and abscisic acid according to the VvRab. Interestingly, and unexpectedly considering transcript expression, western blotting using a monoclonal antibody raised against AtRabA5c (ARA4) showed a specific expression in the exocarp of ripe grape berries, in all seven red and white berry varieties tested. By contrast, no expression was detected in any of the other organs or tissues investigated. This paper contains the first description of Rab GTPases in V. vinifera. The involvement of a specific VvRab in grape berry late development and the potential role of this Rab GTPase are discussed in relation to literature data.

Randomized controlled trial of a community-based intervention on HIV and nutritional outcomes at 6 months among women living with HIV/AIDS in rural India.

OBJECTIVE: To assess the impact of nurse-led Asha (Accredited Social Health Activist)-support behavioral and nutritional intervention among women living with HIV/AIDS (WLH/A) in rural India. DESIGN: Cluster randomized controlled trial. METHODS: Sixteen Primary Health Centers serving WLH/A in Andhra Pradesh were grouped into four regional clusters that were randomly allocated into one of four arms. All four groups included Asha-support and consisted of: Asha-support only (control group); nutrition education; nutrition supplementation; and the combination of supplementation and education. Differences between baseline and 6-month follow-up for key physiological outcomes (BMI, CD4 cell count) were analyzed using factorial mixed models that accounted for geographic clustering. RESULTS: At 6 months, all groups improved CD4 cell count: Asha only [mean difference score (D) = 343.97, standard deviation (SD) = 106.94], nutrition education (D = 356.15, SD = 0.69), nutrition supplement (D = 469.66, SD = 116.0), and nutrition supplement and education (D = 530.82, SD = 128.56). In multivariable models, Asha-support and nutrition, and Asha-support and nutrition supplement interventions demonstrated independent significant improvements in CD4 cell count; the interaction term was significant [estimate = 529.9; 95% confidence interval (CI) 512.0, 547.8; P = 0.006]. BMI also increased for all groups: Asha only (D = 0.95, SD = 0.82), Asha and nutrition education (D = 1.28, SD = 0.53), Asha and nutrition supplement (D = 2.38, SD = 0.60), nutrition supplement, and nutrition supplement and education (D = 2.72, SD = 0.84). Nutrition supplementation and nutrition education demonstrated independent effects on BMI; the interaction term was not significant (estimate = 0.27; 95% CI = 2.5, 2.7; P = 0.80). CONCLUSION: Interventions supported by community workers were efficacious at improving physiological outcomes and may be beneficial at meeting critical healthcare needs of vulnerable WLH/A in India.

Control of Gene Expression in Senescence through Transcriptional Read-Through of Convergent Protein-Coding Genes.

Antisense RNAs are non-coding RNAs that can regulate their corresponding sense RNAs and are generally produced from specific promoters. We uncover here a family of antisense RNAs, named START RNAs, produced during cellular senescence by transcriptional read-through at convergent protein-coding genes. Importantly, START RNAs repress the expression of their corresponding sense RNAs. In proliferative cells, we found that the Pol II elongation rate is limited downstream of TTS at START loci, allowing transcription termination to occur before Pol II reaches the convergent genes, thus preventing antisense RNA production and interference with the expression of the convergent genes. START RNAs are repressed by H2A.Z histone variant, whose local occupancy decreases in senescence. Our results thus uncover a mechanism of gene expression regulation relying on read-through antisense transcript production at convergent genes, underlining the functional importance of chromatin regulation in the control of RNA pol II elongation rate at intergenic regions.

Poly(A) Polymerase and the Nuclear Poly(A) Binding Protein, PABPN1, Coordinate the Splicing and Degradation of a Subset of Human Pre-mRNAs.

Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3'-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing.