Microprocessor, Setx, Xrn2, and Rrp6 co-operate to induce premature termination of transcription by RNAPII.

Transcription elongation is increasingly recognized as an important mechanism of gene regulation. Here, we show that microprocessor controls gene expression in an RNAi-independent manner. Microprocessor orchestrates the recruitment of termination factors Setx and Xrn2, and the 3'-5' exoribonuclease, Rrp6, to initiate RNAPII pausing and premature termination at the HIV-1 promoter through cleavage of the stem-loop RNA, TAR. Rrp6 further processes the cleavage product, which generates a small RNA that is required to mediate potent transcriptional repression and chromatin remodeling at the HIV-1 promoter. Using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq), we identified cellular gene targets whose transcription is modulated by microprocessor. Our study reveals RNAPII pausing and premature termination mediated by the co-operative activity of ribonucleases, Drosha/Dgcr8, Xrn2, and Rrp6, as a regulatory mechanism of RNAPII-dependent transcription elongation.

NF90 interacts with components of RISC and modulates association of Ago2 with mRNA.

BACKGROUND: Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection, and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. RESULTS: Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3' UTRs, NF90 association was increased upon loss of MOV10 and vice versa. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. CONCLUSIONS: These findings reveal that, in the cytoplasm, NF90 can associate with components of RISC such as Ago2 and MOV10. In addition, the data indicate that NF90 and MOV10 may compete for the binding of common target mRNAs, suggesting a role for NF90 in the regulation of RISC-mediated silencing by stabilizing target mRNAs, such as VEGF, during cancer-induced hypoxia.

NF90 modulates processing of a subset of human pri-miRNAs.

MicroRNAs (miRNAs) are predicted to regulate the expression of >60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The double stranded RNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of primary miRNAs (pri-miRNAs). Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA electrophoretic mobility shift assay (EMSA). NF90-bound and downregulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly reduced. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

The Polyvalent Role of NF90 in RNA Biology.

Double-stranded RNA-binding proteins (dsRBPs) are major players in the regulation of gene expression patterns. Among them, Nuclear Factor 90 (NF90) has a plethora of well-known functions in viral infection, transcription, and translation as well as RNA stability and degradation. In addition, NF90 has been identified as a regulator of microRNA (miRNA) maturation by competing with Microprocessor for the binding of pri-miRNAs in the nucleus. NF90 was recently shown to control the biogenesis of a subset of human miRNAs, which ultimately influences, not only the abundance, but also the expression of the host gene and the fate of the mRNA target repertoire. Moreover, recent evidence suggests that NF90 is also involved in RNA-Induced Silencing Complex (RISC)-mediated silencing by binding to target mRNAs and controlling their translation and degradation. Here, we review the many, and growing, functions of NF90 in RNA biology, with a focus on the miRNA pathway and RISC-mediated gene silencing.

Nuclear RNA surveillance complexes silence HIV-1 transcription.

Expression from the HIV-1 LTR can be repressed in a small population of cells, which contributes to the latent reservoir. The factors mediating this repression have not been clearly elucidated. We have identified a network of nuclear RNA surveillance factors that act as effectors of HIV-1 silencing. RRP6, MTR4, ZCCHC8 and ZFC3H1 physically associate with the HIV-1 TAR region and repress transcriptional output and recruitment of RNAPII to the LTR. Knock-down of these factors in J-Lat cells increased the number of GFP-positive cells, with a concomitant increase in histone marks associated with transcriptional activation. Loss of these factors increased HIV-1 expression from infected PBMCs and led to reactivation of HIV-1 from latently infected PBMCs. These findings identify a network of novel transcriptional repressors that control HIV-1 expression and which could open new avenues for therapeutic intervention.

HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP.

HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.

Identification of histone 3 variant 2 interacting factors.

The epigenome is defined as a type of information that can be transmitted independently of the DNA sequence, at the chromatin level, through post-translational modifications present on histone tails. Recent advances in the identification of histone 3 variants suggest a new model of information transmission through deposition of specific histone variants. To date, several non-centromeric histone 3 variants have been identified in mammals. Despite protein sequence similarity, specific deposition complexes have been characterized for both histone 3.1 (H3.1) and histone 3.3 (H3.3), whereas no deposition complex for histone 3.2 (H3.2) has been identified to date. Here, we identified human H3.2 partners by immunopurification of nuclear H3.2 complexes followed by mass spectrometry analysis. Further biochemical analyses highlighted two major complexes associated with H3.2, one containing chromatin associated factor-1 subunits and the other consisting of a subcomplex of mini chromosome maintenance helicases, together with Asf1. The purified complexes could associate with a DNA template in vitro.

Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2.

The p300-CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) involved in the reversible acetylation of various transcriptional regulators, including the tumour suppressor p53. It is implicated in many cellular processes, such as transcription, differentiation, proliferation and apoptosis. We observed that knockdown of PCAF expression in HeLa or U2OS cell lines induces stabilization of the oncoprotein Hdm2, a RING finger E3 ligase primarily known for its role in controlling p53 stability. To investigate the molecular basis of this effect, we examined whether PCAF is involved in Hdm2 ubiquitination. Here, we show that PCAF, in addition to its acetyltransferase activity, possesses an intrinsic ubiquitination activity that is critical for controlling Hdm2 expression levels, and thus p53 functions. Our data highlight a regulatory crosstalk between PCAF and Hdm2 activities, which is likely to have a central role in the subtle control of p53 activity after DNA damage.

PAPγ associates with PAXT nuclear exosome to control the abundance of PROMPT ncRNAs.

Pervasive transcription of the human genome generates an abundance of RNAs that must be processed and degraded. The nuclear RNA exosome is the main RNA degradation machinery in the nucleus. However, nuclear exosome must be recruited to its substrates by targeting complexes, such as NEXT or PAXT. By proteomic analysis, we identify additional subunits of PAXT, including many orthologs of MTREC found in S. pombe. In particular, we show that polyA polymerase gamma (PAPγ) associates with PAXT. Genome-wide mapping of the binding sites of ZFC3H1, RBM27 and PAPγ shows that PAXT is recruited to the TSS of hundreds of genes. Loss of ZFC3H1 abolishes recruitment of PAXT subunits including PAPγ to TSSs and concomitantly increases the abundance of PROMPTs at the same sites. Moreover, PAPγ, as well as MTR4 and ZFC3H1, is implicated in the polyadenylation of PROMPTs. Our results thus provide key insights into the direct targeting of PROMPT ncRNAs by PAXT at their genomic sites.

Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR.

BACKGROUND: Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. RESULTS: Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. CONCLUSION: These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.

Chromatin-associated YTHDC1 coordinates heat-induced reprogramming of gene expression.

Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.

A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter.

The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.

Chromatin-associated MRN complex protects highly transcribing genes from genomic instability.

MRN-MDC1 plays a central role in the DNA damage response (DDR) and repair. Using proteomics of isolated chromatin fragments, we identified DDR factors, such as MDC1, among those highly associating with a genomic locus upon transcriptional activation. Purification of MDC1 in the absence of exogenous DNA damage revealed interactions with factors involved in gene expression and RNA processing, in addition to DDR factors. ChIP-seq showed that MRN subunits, MRE11 and NBS1, colocalized throughout the genome, notably at TSSs and bodies of actively transcribing genes, which was dependent on the RNAPII transcriptional complex rather than transcription per se. Depletion of MRN increased RNAPII abundance at MRE11/NBS1-bound genes. Prolonged MRE11 or NBS1 depletion induced single-nucleotide polymorphisms across actively transcribing MRN target genes. These data suggest that association of MRN with the transcriptional machinery constitutively scans active genes for transcription-induced DNA damage to preserve the integrity of the coding genome.

An NF90/NF110-mediated feedback amplification loop regulates dicer expression and controls ovarian carcinoma progression.

Reduced expression of DICER, a key enzyme in the miRNA pathway, is frequently associated with aggressive, invasive disease, and poor survival in various malignancies. Regulation of DICER expression is, however, poorly understood. Here, we show that NF90/NF110 facilitates DICER expression by controlling the processing of a miRNA, miR-3173, which is embedded in DICER pre-mRNA. As miR-3173 in turn targets NF90, a feedback amplification loop controlling DICER expression is established. In a nude mouse model, NF90 overexpression reduced proliferation of ovarian cancer cells and significantly reduced tumor size and metastasis, whereas overexpression of miR-3173 dramatically increased metastasis in an NF90- and DICER-dependent manner. Clinically, low NF90 expression and high miR-3173-3p expression were found to be independent prognostic markers of poor survival in a cohort of ovarian carcinoma patients. These findings suggest that, by facilitating DICER expression, NF90 can act as a suppressor of ovarian carcinoma.

Premature termination of transcription by RNAP II: the beginning of the end.

Transcription elongation is now recognized as an important mechanism of gene regulation in eukaryotes. A large number of genes undergo an early step in transcription that is rate limiting for expression. Genome-wide studies showing that RNA polymerase II accumulates to high densities near the promoters of many genes has led to the idea that promoter-proximal pausing of transcription is a widespread, rate-limiting step in early elongation. Recent evidence suggests that much of this paused RNA polymerase II is competent for transcription elongation. Here, we discuss recent studies suggesting that RNA polymerase II that accumulates nearby the promoter of a subset of genes is undergoing premature termination of transcription.

The HIV-1 Tat protein stimulates reverse transcription in vitro.

The role of Tat in HIV-1 reverse transcription has been controversial largely because different studies have observed disparate effects of the Tat protein on reverse transcription. Studies of HIV-1 lacking a functional tat gene demonstrated a decrease in reverse transcription efficiency following infection of T-cells however, in vitro recombinant Tat(1-86) has been shown to inhibit RT activity. Here we show that 20-200 nM of both N-terminally histidine-tagged recombinant Tat(1-72) and Tat(1-86) stimulated reverse transcription by HIV-1 reverse transcriptase (RT) in vitro by 2-3 fold. However, both Tat species were efficient inhibitors of RT activity at 400 nM. The lower concentrations of Tat increased reverse transcription efficiency by facilitating multiple rounds of DNA synthesis, and this increase was either not seen or reduced when Tat proteins with multiply-mutated cysteine or basic domains were used. Tat-enhanced reverse transcription occurred in a RNA-independent manner, and required formation of a Tat-RT complex. Pull-down and immunoprecipitation experiments confirmed that Tat could interact with the RT p51 subunit, and mammalian two-hybrid experiments showed interaction between Tat and both the p51 and p66 subunits. Together these results provide evidence that Tat can stimulate reverse transcription through an interaction with RT.

The proteasome regulates HIV-1 transcription by both proteolytic and nonproteolytic mechanisms.

Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter.

Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcriptional silencing and post-integration latency.

HIV-1 gene expression is the major determinant regulating the rate of virus replication and, consequently, AIDS progression. Following primary infection, most infected cells produce virus. However, a small population becomes latently infected and constitutes the viral reservoir. This stable viral reservoir seriously challenges the hope of complete viral eradication. Viewed in this context, it is critical to define the molecular mechanisms involved in the establishment of transcriptional latency and the reactivation of viral expression. We show that Suv39H1, HP1gamma and histone H3Lys9 trimethylation play a major role in chromatin-mediated repression of integrated HIV-1 gene expression. Suv39H1, HP1gamma and histone H3Lys9 trimethylation are reversibly associated with HIV-1 in a transcription-dependent manner. Finally, we show in different cellular models, including PBMCs from HIV-1-infected donors, that HIV-1 reactivation could be achieved after HP1gamma RNA interference.

Requirement for SWI/SNF chromatin-remodeling complex in Tat-mediated activation of the HIV-1 promoter.

Activation of the human immunodeficiency virus type-1 (HIV-1) promoter in infected cells requires the sequential recruitment of several cellular factors to facilitate the formation of a processive elongation complex. The nucleosomal reorganization of the HIV-1 long terminal repeat (LTR) observed upon Tat stimulation suggests that chromatin-remodeling complexes could play a role during this process. Here, we reported that Tat interacts directly with Brm, a DNA-dependent ATPase subunit of the SWI/SNF chromatin-remodeling complex, to activate the HIV-1 LTR. Inhibition of Brm via small interfering RNAs impaired Tat-mediated transactivation of an integrated HIV-1 promoter. Furthermore, Brm is recruited in vivo to the HIV-1 LTR in a Tat-dependent manner. Interestingly, we found that Tat/Brm interaction is regulated by Tat lysine 50 acetylation. These data show the requirement of Tat-mediated recruitment of SWI/SNF chromatin-remodeling complex to HIV-1 promoter in the activation of the LTR.