Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination.

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases.

Integrator regulates transcriptional initiation and pause release following activation.

In unicellular organisms, initiation is the rate-limiting step in transcription; in metazoan organisms, the transition from initiation to productive elongation is also important. Here, we show that the RNA polymerase II (RNAPII)-associated multiprotein complex, Integrator, plays a critical role in both initiation and the release of paused RNAPII at immediate early genes (IEGs) following transcriptional activation by epidermal growth factor (EGF) in human cells. Integrator is recruited to the IEGs in a signal-dependent manner and is required to engage and recruit the super elongation complex (SEC) to EGF-responsive genes to allow release of paused RNAPII and productive transcription elongation.

Molecular basis for the interaction between Integrator subunits IntS9 and IntS11 and its functional importance.

The metazoan Integrator complex (INT) has important functions in the 3'-end processing of noncoding RNAs, including the uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA), and in the transcription of coding genes by RNA polymerase II. The INT contains at least 14 subunits, but its molecular mechanism of action is poorly understood, because currently there is little structural information about its subunits. The endonuclease activity of INT is mediated by its subunit 11 (IntS11), which belongs to the metallo-ß-lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with Integrator complex subunit 9 (IntS9) through their C-terminal domains (CTDs). Here, we report the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and detailed, structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded ß-sheet with four strands from IntS9 and five from IntS11. Highly conserved residues are located in the extensive interface between the two CTDs. Yeast two-hybrid assays and coimmunoprecipitation experiments confirm the structural observations on the complex. Functional studies demonstrate that the IntS9-IntS11 interaction is crucial for the role of INT in snRNA 3'-end processing.

Correction: Human mutations in integrator complex subunits link transcriptome integrity to brain development.

[This corrects the article DOI: 10.1371/journal.pgen.1006809.].

Human mutations in integrator complex subunits link transcriptome integrity to brain development.

Integrator is an RNA polymerase II (RNAPII)-associated complex that was recently identified to have a broad role in both RNA processing and transcription regulation. Importantly, its role in human development and disease is so far largely unexplored. Here, we provide evidence that biallelic Integrator Complex Subunit 1 (INTS1) and Subunit 8 (INTS8) gene mutations are associated with rare recessive human neurodevelopmental syndromes. Three unrelated individuals of Dutch ancestry showed the same homozygous truncating INTS1 mutation. Three siblings harboured compound heterozygous INTS8 mutations. Shared features by these six individuals are severe neurodevelopmental delay and a distinctive appearance. The INTS8 family in addition presented with neuronal migration defects (periventricular nodular heterotopia). We show that the first INTS8 mutation, a nine base-pair deletion, leads to a protein that disrupts INT complex stability, while the second missense mutation introduces an alternative splice site leading to an unstable messenger. Cells from patients with INTS8 mutations show increased levels of unprocessed UsnRNA, compatible with the INT function in the 3'-end maturation of UsnRNA, and display significant disruptions in gene expression and RNA processing. Finally, the introduction of the INTS8 deletion mutation in P19 cells using genome editing alters gene expression throughout the course of retinoic acid-induced neural differentiation. Altogether, our results confirm the essential role of Integrator to transcriptome integrity and point to the requirement of the Integrator complex in human brain development.

Integrator: surprisingly diverse functions in gene expression.

The discovery of the metazoan-specific Integrator (INT) complex represented a breakthrough in our understanding of noncoding U-rich small nuclear RNA (UsnRNA) maturation and has triggered a reevaluation of their biosynthesis mechanism. In the decade since, significant progress has been made in understanding the details of its recruitment, specificity, and assembly. While some discrepancies remain on how it interacts with the C-terminal domain (CTD) of the RNA polymerase II (RNAPII) and the details of its recruitment to UsnRNA genes, preliminary models have emerged. Recent provocative studies now implicate INT in the regulation of protein-coding gene transcription initiation and RNAPII pause-release, thereby broadening the scope of INT functions in gene expression regulation. We discuss the implications of these findings while putting them into the context of what is understood about INT function at UsnRNA genes.

Genome-wide analysis reveals a role for BRCA1 and PALB2 in transcriptional co-activation.

Breast and ovarian cancer susceptibility genes BRCA1 and PALB2 have enigmatic roles in cellular growth and mammalian development. While these genes are essential for growth during early developmental programs, inactivation later in adulthood results in increased growth and formation of tumors, leading to their designation as tumor suppressors. We performed genome-wide analysis assessing their chromatin residence and gene expression responsiveness using high-throughput sequencing in breast epithelial cells. We found an intimate association between BRCA1 and PALB2 chromatin residence and genes displaying high transcriptional activity. Moreover, our experiments revealed a critical role for BRCA1 and, to a smaller degree, PALB2 in transcriptional responsiveness to NF-κB, a crucial mediator of growth and inflammatory response during development and cancer. Importantly, we also uncovered a vital role for BRCA1 and PALB2 in response to retinoic acid (RA), a growth inhibitory signal in breast cancer cells, which may constitute the basis for their tumor suppressor activity. Taken together, our results highlight an important role for these breast cancer proteins in the regulation of diverse growth regulatory pathways.

CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells.

The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT.

MicroRNA silencing through RISC recruitment of eIF6.

MicroRNAs (miRNAs) are a class of small RNAs that act post-transcriptionally to regulate messenger RNA stability and translation. To elucidate how miRNAs mediate their repressive effects, we performed biochemical and functional assays to identify new factors in the miRNA pathway. Here we show that human RISC (RNA-induced silencing complex) associates with a multiprotein complex containing MOV10--which is the homologue of Drosophila translational repressor Armitage--and proteins of the 60S ribosome subunit. Notably, this complex contains the anti-association factor eIF6 (also called ITGB4BP or p27BBP), a ribosome inhibitory protein known to prevent productive assembly of the 80S ribosome. Depletion of eIF6 in human cells specifically abrogates miRNA-mediated regulation of target protein and mRNA levels. Similarly, depletion of eIF6 in Caenorhabditis elegans diminishes lin-4 miRNA-mediated repression of the endogenous LIN-14 and LIN-28 target protein and mRNA levels. These results uncover an evolutionarily conserved function of the ribosome anti-association factor eIF6 in miRNA-mediated post-transcriptional silencing.

Ets-1 p51 and p42 isoforms differentially modulate Stromelysin-1 promoter according to induced DNA bend orientation.

The Stromelysin-1 gene promoter contains a palindrome of two Ets-binding sites (EBS) that bind the p51 and p42 isoforms of the human Ets-1-transcription factor. A previous study established that full gene transactivation is associated with a ternary complex consisting of two p51 bound to the two EBS on the promoter. p42, only able to bind one of the two EBS, induces only very weak activity. Here, we investigate the mechanism by which the Stromelysin-1 promoter discriminates between p51 and p42. The differential stoichiometry of the two Ets-1 isoforms arises from the Stromelysin-1 EBS palindrome. The ternary complex requires the presence of two inhibitory domains flanking the DNA-binding domain and the ability to form an intramolecular autoinhibition module. Most importantly, the p51-ternary and the p42-binary complexes induce DNA curvatures with opposite orientations. These results establish that differential DNA bending, via p51 and p42 differential binding, is correlated with the Stromelysin-1 promoter activation process.

Huntington's disease protein contributes to RNA-mediated gene silencing through association with Argonaute and P bodies.

Huntington's disease is a dominant autosomal neurodegenerative disorder caused by an expansion of polyglutamines in the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function, we purified epitope-tagged Htt and identified Argonaute as associated proteins. Colocalization studies demonstrated Htt and Ago2 to be present in P bodies, and depletion of Htt showed compromised RNA-mediated gene silencing. Mouse striatal cells expressing mutant Htt showed fewer P bodies and reduced reporter gene silencing activity compared with wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes.

Ets-1 binds cooperatively to the palindromic Ets-binding sites in the p53 promoter.

Due to its autoinhibition for DNA binding, the Ets-1 transcription factor must interact with partners to enhance its affinity for DNA. In a study on the stromelysin-1 promoter, we showed that Ets-1 binds cooperatively to two Ets-binding sites (EBS) organized in palindrome, thereby circumventing the need for a binding partner to counteract autoinhibition. This leads to the formation of an Ets-1-DNA-Ets-1 ternary complex necessary for promoter activation. Here we show that Ets-1 also binds cooperatively to the EBS palindrome of the human p53 promoter, despite the presence of a degenerate EBS to which Ets-1 cannot otherwise bind. Transcriptional transactivation through this palindrome fully correlates to Ets-1 binding. Thus, the cooperative binding model that we initially proposed for the stromelysin-1 promoter may be a general mechanism of Ets-1 binding to palindromic EBS separated by 4bp and a way to counteract binding site degeneracy.

Composition of the Survival Motor Neuron (SMN) Complex in Drosophila melanogaster.

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated its composition in Drosophila melanogaster Using transgenic flies that exclusively express Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

Efficient system for biotinylated recombinant Ets-1 production in Escherichia coli: a useful tool for studying interactions between Ets-1 and its partners.

Identification of Ets-1 interaction partners is critical for understanding its properties. Ets-1 DNA-binding is governed by an intramolecular mechanism called autoinhibition. Ets-1 increases its DNA-binding affinity by counteracting autoinhibition through binding either to a particular organization of Ets binding sites (EBS) in palindrome, as in the Stromelysin-1 promoter, or to EBS adjacent to DNA-binding sites of its partners by combinatorial interactions, as in the Collagenase-1 promoter. Identification of new Ets-1 interaction partners should allow the identification of new functions for this transcription factor. To this end, we fused a biotin tag to Ets-1 protein in order to copurify it and its partners by affinity. For the first time, we cloned, produced in Escherichia coli and purified a biotinylated recombinant Ets-1 protein using the T7-Impact system (New England Biolabs), adapted to induce biotinylation. Nearly 100% biotinylation was attained without altering Ets-1 properties. Biotinylated Ets-1 bound to and transactivated the Stromelysin-1 promoter the same way as native Ets-1 did. It also conserved interactions with known Ets-1 partners such as c-Jun, Erk-2 and Runx-1. In addition, streptavidin pull-down and surface plasmon resonance assays demonstrated that biotinylated Ets-1 is a useful tool for qualitative and quantitative studies of Ets-1 interaction with its partners.

3' UTR shortening represses tumor-suppressor genes in trans by disrupting ceRNA crosstalk.

Widespread mRNA 3' UTR shortening through alternative polyadenylation 1 promotes tumor growth in vivo 2 . A prevailing hypothesis is that it induces proto-oncogene expression in cis through escaping microRNA-mediated repression. Here we report a surprising enrichment of 3'UTR shortening among transcripts that are predicted to act as competing-endogenous RNAs (ceRNAs) for tumor-suppressor genes. Our model-based analysis of the trans effect of 3' UTR shortening (MAT3UTR) reveals a significant role in altering ceRNA expression. MAT3UTR predicts many trans-targets of 3' UTR shortening, including PTEN, a crucial tumor-suppressor gene 3 involved in ceRNA crosstalk 4 with nine 3'UTR-shortening genes, including EPS15 and NFIA. Knockdown of NUDT21, a master 3' UTR-shortening regulator 2 , represses tumor-suppressor genes such as PHF6 and LARP1 in trans in a miRNA-dependent manner. Together, the results of our analysis suggest a major role of 3' UTR shortening in repressing tumor-suppressor genes in trans by disrupting ceRNA crosstalk, rather than inducing proto-oncogenes in cis.

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCFSlmb degron.

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1 Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

Linking transcriptional elongation and messenger RNA export to metastatic breast cancers.

The biochemical pathways that are disrupted in the genesis of sporadic breast cancers remain unclear. Moreover, the present prognosticating markers used to determine the prognosis of node-negative patient leads to probabilistic results, and the eventual clinical course is far from certain. Here we identified the human TREX complex, a multiprotein complex that links transcription elongation to mRNA transport, as culprit of aggressive human breast cancers. We show that whereas p84N5 (called hTREX84) is expressed at very low levels in normal breast epithelial cells, it is highly expressed in breast tumors. Importantly, hTREX84 expression correlates with tumor size and the metastatic state of the tumor progression. Reduction of hTREX84 levels in breast cancer cell lines by small interfering RNA result in inhibition of cellular proliferation and abrogation of mRNA export. These results not only identify hTREX84 as a prognosticator of breast cancer but also delineate human TREX complex as a target for therapeutic drugs against breast cancer.

Characterization and functional analysis of the p42Ets-1 variant of the mouse Ets-1 transcription factor.

We have identified the mouse exon VII splice variant of the Ets-1 transcription factor. The variant is expressed in all cell lines which express ets-1, at lower levels, it is also expressed in the mouse embryo in vivo. The corresponding protein, p42Ets-1, is a transcription factor as it is able to bind to specific DNA sequences and to transactivate a bona fide ETS reporter vector. A comparison of optimal DNA-binding sites shows that p42Ets-1 binds to more various DNA sequences than p51Ets-1; p42Ets-1 recognizes the same optimal consensus sequence as p51Ets-1, but also many variations of it, mainly at base -1, which is located just prior to the GGAA/T core sequence. The binding differences were quantified by surface plasmon resonance analyses and the protein region responsible for the differences in DNA sequence recognition located in the Val280-Glu302 fragment, which is encoded by exon VII. The specific DNA-binding properties of each isoform translates into clear differences in activity, p42Ets-1 transactivates the natural VE-cadherin gene promoter through both ETS-binding site (EBS)2 and EBS4 whereas p51Ets-1 is mainly active on EBS4. Altogether, our data suggest that p42Ets-1 acts as a distinct transcription factor from p51Ets-1.

Non-mRNA 3' end formation: how the other half lives.

The release of nascent RNA from transcribing RNA polymerase complexes is required for all further functions carried out by RNA molecules. The elements and processing machinery involved in 3' end formation therefore represent key determinants in the biogenesis and accumulation of cellular RNA. While these factors have been well-characterized for messenger RNA, recent work has elucidated analogous pathways for the 3' end formation of other important cellular RNA. Here, we discuss four specific cases of non-mRNA 3' end formation-metazoan small nuclear RNA, Saccharomyces cerevisiae small nuclear RNA, Schizosaccharomyces pombe telomerase RNA, and the mammalian MALAT1 large noncoding RNA-as models of alternative mechanisms to generate RNA 3' ends. Comparison of these disparate processing pathways reveals an emerging theme of evolutionary ingenuity. In some instances, evidence for the creation of a dedicated processing complex exists; while in others, components are utilized from the existing RNA processing machinery and modified to custom fit the unique needs of the RNA substrate. Regardless of the details of how non-mRNA 3' ends are formed, the lengths to which biological systems will go to release nascent transcripts from their DNA templates are fundamental for cell survival.

Requirement for SNAPC1 in transcriptional responsiveness to diverse extracellular signals.

Initiation of transcription of RNA polymerase II (RNAPII)-dependent genes requires the participation of a host of basal transcription factors. Among genes requiring RNAPII for transcription, small nuclear RNAs (snRNAs) display a further requirement for a factor known as snRNA-activating protein complex (SNAPc). The scope of the biological function of SNAPc and its requirement for transcription of protein-coding genes has not been elucidated. To determine the genome-wide occupancy of SNAPc, we performed chromatin immunoprecipitation followed by high-throughput sequencing using antibodies against SNAPC4 and SNAPC1 subunits. Interestingly, while SNAPC4 occupancy was limited to snRNA genes, SNAPC1 chromatin residence extended beyond snRNA genes to include a large number of transcriptionally active protein-coding genes. Notably, SNAPC1 occupancy on highly active genes mirrored that of elongating RNAPII extending through the bodies and 3' ends of protein-coding genes. Inhibition of transcriptional elongation resulted in the loss of SNAPC1 from the 3' ends of genes, reflecting a functional association between SNAPC1 and elongating RNAPII. Importantly, while depletion of SNAPC1 had a small effect on basal transcription, it diminished the transcriptional responsiveness of a large number of genes to two distinct extracellular stimuli, epidermal growth factor (EGF) and retinoic acid (RA). These results highlight a role for SNAPC1 as a general transcriptional coactivator that functions through elongating RNAPII.