Dawn O'Reilly

Serine-7 of the RNA Polymerase II CTD Is Specifically Required for snRNA Gene Expression

RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene–specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type–specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.

A single serine residue at position 375 of VP16 is critical for complex assembly with Oct‐1 and HCF and is a target of phosphorylation by casein kinase II

We show that VP16 is phosphorylated by cellular kinases in vivo and in vitro and map the major sites of phosphorylation to be on serines towards the C–terminus, downstream of position 370 in both cases. Deletion of the acidic activation domain had no effect on phosphorylation, refining the sites to between position 370 and 411. Within VP16, the C‐terminal boundary for complex formation with Oct‐1 and HCF lies at position 388, and between 370 and 388 lies one serine, at position 375. This is a consensus casein kinase II (CKII) site and, using purified wild‐type and mutant proteins, we show that it is the main CKII site in the body of the N‐terminal complex‐forming region. This site is also phosphorylated in nuclear extracts. Although other sites, mainly Ser411, are also phosphorylated by nuclear kinase(s), the single substitution of Ser375 to alanine abolishes CKII phosphorylation in vitro and virtually eliminates complex formation. This serine lies in a surface‐exposed region of VP16 and, although complex formation is disrupted, other activities of the mutant are unaffected. Ser375 is also required in vivo where substitution to alanine abolishes transactivation, while replacement with threonine restores normal levels of activity.

Chromatin Structure Is Implicated in “Late” Elongation Checkpoints on the U2 snRNA and β-Actin Genes

ABSTRACT The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the β-actin gene, where a “late” P-TEFb-dependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and β-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes.

Splicing of many human genes involves sites embedded within introns.

The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome.

Herpes Simplex Virus 1 (HSV-1) ICP22 Protein Directly Interacts with Cyclin-Dependent Kinase (CDK)9 to Inhibit RNA Polymerase II Transcription Elongation

The Herpes Simplex Virus 1 (HSV-1)-encoded ICP22 protein plays an important role in viral infection and affects expression of host cell genes. ICP22 is known to reduce the global level of serine (Ser)2 phosphorylation of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptapeptide repeats comprising the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II. Accordingly, ICP22 is thought to associate with and inhibit the activity of the positive-transcription elongation factor b (P-TEFb) pol II CTD Ser2 kinase. We show here that ICP22 causes loss of CTD Ser2 phosphorylation from pol II engaged in transcription of protein-coding genes following ectopic expression in HeLa cells and that recombinant ICP22 interacts with the CDK9 subunit of recombinant P-TEFb. ICP22 also interacts with pol II in vitro. Residues 193 to 256 of ICP22 are sufficient for interaction with CDK9 and inhibition of pol II CTD Ser2 phosphorylation but do not interact with pol II. These results indicate that discrete regions of ICP22 interact with either CDK9 or pol II and that ICP22 interacts directly with CDK9 to inhibit expression of host cell genes.

Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease

The U1 small nuclear (sn)RNA (U1) is a multifunctional ncRNA, known for its pivotal role in pre-mRNA splicing and regulation of RNA 3′ end processing events. We recently demonstrated that a new class of human U1-like snRNAs, the variant (v)U1 snRNAs (vU1s), also participate in pre-mRNA processing events. In this study, we show that several human vU1 genes are specifically upregulated in stem cells and participate in the regulation of cell fate decisions. Significantly, ectopic expression of vU1 genes in human skin fibroblasts leads to increases in levels of key pluripotent stem cell mRNA markers, including NANOG and SOX2. These results reveal an important role for vU1s in the control of key regulatory networks orchestrating the transitions between stem cell maintenance and differentiation. Moreover, vU1 expression varies inversely with U1 expression during differentiation and cell re-programming and this pattern of expression is specifically de-regulated in iPSC-derived motor neurons from Spinal Muscular Atrophy (SMA) type 1 patients. Accordingly, we suggest that an imbalance in the vU1/U1 ratio, rather than an overall reduction in Uridyl-rich (U)-snRNAs, may contribute to the specific neuromuscular disease phenotype associated with SMA.

Variant snRNPs: New players within the spliceosome system

ABSTRACT Much evidence is now accumulating that, in addition to their general role in splicing, the components of the core splicing machinery have extensive regulatory potential. In particular, recent evidence has demonstrated that de-regulation of these factors cause the highest extent of alternative splicing changes compared to de-regulation of the classical splicing regulators. This lack of a general inhibition of splicing resonates the differential splicing effects observed in different disease pathologies associated with specific mutations targeting core spliceosomal components. In this review we will summarize what is currently known regarding the involvement of core spliceosomal U-snRNP complexes in perturbed tissue development and human diseases and argue for the existence of a compensatory mechanism enabling cells to cope with drastic perturbations in core splicing components. This system maintains the correct balance of spliceosomal snRNPs through differential expression of variant (v)U-snRNPs.

Analysis of novel missense ATR mutations reveals new splicing defects underlying Seckel syndrome

Ataxia Telangiectasia and Rad3 related (ATR) is one of the main regulators of the DNA damage response. It coordinates cell cycle checkpoint activation, replication fork stability, restart and origin firing to maintain genome integrity. Mutations of the ATR gene have been reported in Seckel patients, who suffer from a rare genetic disease characterized by severe microcephaly and growth retardation. Here, we report the case of a Seckel patient with compound heterozygous mutations in ATR. One allele has an intronic mutation affecting splicing of neighboring exons, the other an exonic missense mutation, producing the variant p.Lys1665Asn, of unknown pathogenicity. We have modeled this novel missense mutation, as well as a previously described missense mutation p.Met1159Ile, and assessed their effect on ATR function. Interestingly, our data indicate that both missense mutations have no direct effect on protein function, but rather result in defective ATR splicing. These results emphasize the importance of splicing mutations in Seckel Syndrome.