Noncoding RNA 7SK Research Articles

BCL11b interacts with RNA and proteins involved in RNA processing and developmental diseases

BCL11b is a transcription regulator and a tumor suppressor involved in lymphomagenesis, central nervous system (CNS) and immune system developments. BCL11b favors persistence of HIV latency and contributes to control cell cycle, differentiation and apoptosis in multiple organisms and cell models. Although BCL11b recruits the non-coding RNA 7SK and epigenetic enzymes to regulate gene expression, BCL11b-associated ribonucleoprotein complexes are unknown. Thanks to CLIP-seq and quantitative LC-MS/MS mass spectrometry approaches complemented with systems biology validations, we show that BCL11b interacts with RNA splicing and non-sense-mediated decay proteins, including FUS, SMN1, UPF1 and Drosha, which may contribute in isoform selection of protein-coding RNA isoforms from noncoding-RNAs isoforms (retained introns or nonsense mediated RNA). Interestingly, BCL11b binds to RNA transcripts and proteins encoded by the same genes (FUS, ESWR1, CHD and Tubulin). Our study highlights that BCL11b targets RNA processing and splicing proteins, and RNAs that implicate cell cycle, development, neurodegenerative, and cancer pathways. These findings will help future mechanistic understanding of developmental disorders. ImportanceBCL11b-protein and RNA interactomes reveal BLC11b association with specific nucleoprotein complexes involved in the regulation of genes expression.BCL11b interacts with RNA processing and splicing proteins.

Deficiency of m 6 A RNA methylation promotes ZBP1-mediated cell death.

m 6 A RNA methylation suppresses the immunostimulatory potential of endogenous RNA. Deficiency of m 6 A provokes inflammatory responses and cell death, but the underlying mechanisms remain elusive. Here we showed that the noncoding RNA 7SK gains immunostimulatory potential upon m 6 A depletion and subsequently activates the RIG-I/MAVS axis to spark interferon (IFN) signaling cascades. Concomitant excess of IFN and m 6 A deficiency synergistically facilitate the formation of RNA G-quadruplexes (rG4) to promote ZBP1-mediated necroptotic cell death. Collectively, our findings delineate a hitherto uncharacterized mechanism that links m 6 A dysregulation with ZBP1 activity in triggering inflammatory cell death.

7SK truncation at 128-179 nt suppresses embryonic stem cell proliferation in vitro by downregulating CDC6

To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD). ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting. We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6. 7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.

Keeping the balance: The noncoding RNA 7SK as a master regulator for neuron development and function.

The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P-TEFb. Release of P-TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P-TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly important for neuron development and maintenance and it can thus be hypothesized that 7SK is at the center of many signaling pathways contributing to neuron function. 7SK activates neuronal gene expression programs that are key for terminal differentiation of neurons. Proteomics studies revealed a complex protein interactome of 7SK that includes several RNA-binding proteins. Some of these novel 7SK subcomplexes exert non-canonical cytosolic functions in neurons by regulating axonal mRNA transport and fine-tuning spliceosome production in response to transcription alterations. Thus, a picture emerges according to which 7SK acts as a multi-functional RNA scaffold that is integral for neuron homeostasis.

7SK Acts as an Anti-tumor Factor in Tongue Squamous Cell Carcinoma.

Increasing evidence has shown the mechanistic insights about non-coding RNA 7SK in controlling the transcription. However, the biological function and mechanism of 7SK in cancer are largely unclear. Here, we show that 7SK is down-regulated in human tongue squamous carcinoma (TSCC) and acts as a TSCC suppressor through multiple cell-based assays including a migration assay and a xenograft mouse model. The expression level of 7SK was negatively correlated with the size of tumors in the 73 in-house collected TSCC patients. Through combined analysis of 7SK knockdown of RNA-Seq and available published 7SK ChIRP-seq data, we identified 27 of 7SK-regulated genes that were involved in tumor regulation and whose upstream regulatory regions were bound by 7SK. Motif analysis showed that the regulatory sequences of these genes were enriched for transcription factors FOXJ3 and THRA, suggesting a potential involvement of FOXJ3 and THRA in 7SK-regulated genes. Interestingly, the augmented level of FOXJ3 in TSCC patients and previous reports on THRA in other cancers have suggested that these two factors may promote TSCC progression. In support of this idea, we found that 21 out of 27 aforementioned 7SK-associated genes were regulated by FOXJ3 and THRA, and 12 of them were oppositely regulated by 7SK and FOXJ3/THRA. We also found that FOXJ3 and THRA dramatically promoted migration in SCC15 cells. Collectively, we identified 7SK as an antitumor factor and suggested a potential involvement of FOXJ3 and THRA in 7SK-mediated TSCC progression.

Alazami syndrome in an Afghani girl: A case report and review of literature

Purpose: Alazami syndrome is a rare autosomal recessive disorder with core phenotypic manifestations of short stature, mild facial dysmorphism, and global developmental delay evolving to severe intellectual disability. Homozygous loss-of-function mutations in LARP7 gene, which encodes a chaperone protein of the noncoding RNA 7SK, have been detected in patients with Alazami syndrome. Since its fi rst description in 2012, only six families with Alazami syndrome have been reported to date. This case is reported to expand the phenotypic description to include small kidneys.

Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells

Sox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role for Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at gene regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interacts with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but could rather regulate other processes in the nucleoplasm.

A splice-altering variant in LARP7 gene leads to exon exclusion

The LARP7 gene, is a member of the LARP family and is implicated in the formation and stability of the non-coding RNA 7SK, inhibition of P-TEFb-dependent transcription and telomerase assembly. All LARP7 mutations which have been identified so far are frameshift mutations, with the exception of one in an Iranian family which was a substitution in a splice donor site. In this report, we showed that LARP7 splice donor site mutation results in aberrant splicing and we also described how Drosophila Larp7 is required for the formation of long-term behavioral memory.

HnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons

Disturbed RNA processing and subcellular transport contribute to the pathomechanisms of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. RNA-binding proteins are involved in these processes, but the mechanisms by which they regulate the subcellular diversity of transcriptomes, particularly in axons, are not understood. Heterogeneous nuclear ribonucleoprotein R (hnRNP R) interacts with several proteins involved in motoneuron diseases. It is located in axons of developing motoneurons, and its depletion causes defects in axon growth. Here, we used individual nucleotide-resolution cross-linking and immunoprecipitation (iCLIP) to determine the RNA interactome of hnRNP R in motoneurons. We identified ∼3,500 RNA targets, predominantly with functions in synaptic transmission and axon guidance. Among the RNA targets identified by iCLIP, the noncoding RNA 7SK was the top interactor of hnRNP R. We detected 7SK in the nucleus and also in the cytosol of motoneurons. In axons, 7SK localized in close proximity to hnRNP R, and depletion of hnRNP R reduced axonal 7SK. Furthermore, suppression of 7SK led to defective axon growth that was accompanied by axonal transcriptome alterations similar to those caused by hnRNP R depletion. Using a series of 7SK-deletion mutants, we show that the function of 7SK in axon elongation depends on its interaction with hnRNP R but not with the PTEF-B complex involved in transcriptional regulation. These results propose a role for 7SK as an essential interactor of hnRNP R to regulate its function in axon maintenance.

Unraveling the conformational determinants of LARP7 and 7SK small nuclear RNA by theoretical approaches.

LARP7, a member of the La-related proteins (LARPs), shares a conserved La module comprising the La-motif (LAM) and the RNA-recognition motif (RRM1), binding exclusively to the non-coding RNA 7SK. LARP7 is a component of the small nuclear ribonucleoprotein (7SKsnRNP) required for the stability and function of the RNA, and implicated in the transcription termination and regulation of translation. In the current work, molecular dynamics simulations were employed to investigate the recently determined crystal structures of the La module of LARP7 in complexs with a stretch of uridines at the 3'-end of 7SK in the presence and absence of RNA and two different mutants. The structural stabilities of the four systems provided by the simulations are consistent with the experimental data. Principal component analysis (PCA) and free energy landscape (FEL) were used to explore the dominant motions and the functional dynamics between the two ends of the superhelical structures in both RNA-bound and RNA-free systems. The final values of the intramolecular angle formed by the Cα atoms of Arg30, Lys53 and Pro189 are ∼96° and 125° for the RNA-bound and RNA-free systems, highlighting the importance of the binding of the 3'-end of RNA 7SK for system stability. The dynamic cross-correlation maps (DCCM) were utilized to evaluate the conformational changes in different mutants, and small values were found around the residues 29-50 and 100-120 in the F168A system, whereas large values were found around the residues 120-160 and 170-189 in the E130A system. The time evolutions of the hydrogen-bond distances of the terminal uridine U-1 and Asp54 and that of the penultimate residue U-2 and Gln41 were monitored to compare their conformational changes, and the results suggest that the E130A mutant may have an important effect on the RNA binding, which is consistent with site-directed mutagenesis. This study provides some new insights into the understanding of the recognition mechanism between the La module of LARP7 and RNA 7SK.

Intermolecular recognition of the non-coding RNA 7SK and HEXIM protein in perspective.

A 7SKsnRNP complex, comprising the non-coding RNA 7SK and proteins MePCE and LARP7, participates in the regulation of the transcription elongation by RNA-polymerase II in higher eukaryotes. Binding of a HEXIM protein triggers the inhibition of the kinase complex P-TEFb, a key actor of the switch from paused transcription to elongation. The present paper reviews what is known about the specific recognition of the 7SK RNA by the HEXIM protein. HEXIM uses an arginine-rich motif (ARM) peptide to bind one specific site in the 5'-hairpin of the 7SK RNA. Since HEXIM forms a dimer, what happens with the second ARM impacts the assembly symmetry. In order to help sort through possible models, a combination of native mass spectrometry and electrophoretic mobility shift assays was used. It provides evidence that only one ARM of the HEXIM dimer is directly binding to the RNA hairpin and that another sequence downstream of the ARM participates in a second binding event allowing the other monomer of HEXIM to bind the RNA.

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3′-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3′ hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5′-end with the foot of the 3′ hairpin and has thus functional implications.

Ars2 Promotes Proper Replication-Dependent Histone mRNA 3′ End Formation

Ars2 is a component of the nuclear cap-binding complex that contributes to microRNA biogenesis and is required for cellular proliferation. Here, we expand on the repertoire of Ars2-dependent microRNAs and determine that Ars2 regulates a number of mRNAs, the largest defined subset of which code for histones. Histone mRNAs are unique among mammalian mRNAs because they are not normally polyadenylated but, rather, are cleaved following a 3' stem loop. A significant reduction in correctly processed histone mRNAs was observed following Ars2 depletion, concurrent with an increase in polyadenylated histone transcripts. Furthermore, Ars2 physically associated with histone mRNAs and the noncoding RNA 7SK. Knockdown of 7SK led to an enhanced ratio of cleaved to polyadenylated histone transcripts, an effect dependent on Ars2. Together, the data demonstrate that Ars2 contributes to histone mRNA 3' end formation and expression and these functional properties of Ars2 are negatively regulated by interaction with 7SK RNA.