Regulation of the RNAPII Pool Is Integral to the DNA Damage Response.

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.

RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit.

Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here, we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced co-transcriptional splicing. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.

Strand-specific, high-resolution mapping of modified RNA polymerase II.

Reversible modification of the RNAPII C-terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand-specific, nucleotide-resolution information, and we used a machine learning-based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3', with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3' ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein-coding genes and persisted throughout exon 1 of intron-containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein-coding genes.

H19 antisense RNA can up-regulate Igf2 transcription by activation of a novel promoter in mouse myoblasts.

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.

A yeast exosome cofactor, Mpp6, functions in RNA surveillance and in the degradation of noncoding RNA transcripts.

A genome-wide screen for synthetic lethal (SL) interactions with loss of the nuclear exosome cofactors Rrp47/Lrp1 or Air1 identified 3'-->5' exonucleases, the THO complex required for mRNP assembly, and Ynr024w (Mpp6). SL interactions with mpp6Delta were confirmed for rrp47Delta and nuclear exosome component Rrp6. The results of bioinformatic analyses revealed homology between Mpp6 and a human exosome cofactor, underlining the high conservation of the RNA surveillance system. Mpp6 is an RNA binding protein that physically associates with the exosome and was localized throughout the nucleus. The results of functional analyses demonstrated roles for Mpp6 in the surveillance of both pre-rRNA and pre-mRNAs and in the degradation of "cryptic" noncoding RNAs (ncRNAs) derived from intergenic regions and the ribosomal DNA spacer heterochromatin. Strikingly, these ncRNAs are also targeted by other exosome cofactors, including Rrp47, the TRAMP complex (which includes Air1), and the Nrd1/Nab3 complex, and are degraded by both Rrp6 and the core exosome. Heterochromatic transcripts and other ncRNAs are characterized by very rapid degradation, and we predict that functional redundancy is an important feature of ncRNA metabolism.

Inactivation of cleavage factor I components Rna14p and Rna15p induces sequestration of small nucleolar ribonucleoproteins at discrete sites in the nucleus.

Small nucleolar RNAs (snoRNAs) associate with specific proteins forming small nucleolar ribonucleoprotein (snoRNP) particles, which are essential for ribosome biogenesis. The snoRNAs are transcribed, processed, and assembled in snoRNPs in the nucleoplasm. Mature particles are then transported to the nucleolus. In yeast, 3'-end maturation of snoRNAs involves the activity of Rnt1p endonuclease and cleavage factor IA (CFIA). We report that after inhibition of CFIA components Rna14p and Rna15p, the snoRNP proteins Nop1p, Nop58p, and Gar1p delocalize from the nucleolus and accumulate in discrete nucleoplasmic foci. The U14 snoRNA, but not U3 snoRNA, similarly redistributes from the nucleolus to the nucleoplasmic foci. Simultaneous depletion of either Rna14p or Rna15p and the nuclear exosome component Rrp6p induces accumulation of poly(A)(+) RNA at the snoRNP-containing foci. We propose that the foci detected after CFIA inactivation correspond to quality control centers in the nucleoplasm.

Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of polyadenylated RNAs in a discrete domain within the nucleolus.

Recent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)(+) RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)(+) RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.

A nuclear surveillance pathway for mRNAs with defective polyadenylation.

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3'-5' exonucleases, accumulation of poly(A)+ mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6delta. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3'-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

Eosinophil-rich CD30+ lymphoproliferative disorder of the oral mucosa. A form of "traumatic eosinophilic granuloma".

We describe 3 patients who had oral mucosal lesions with features of traumatic eosinophilic granuloma (TEG) and containing CD30+ atypical cells. In 1 patient, the oral lesion was followed by skin nodules. All lesions were evaluated histologically, by immunohistochemical analysis, and by polymerase chain reaction (PCR) analysis of the T-cell receptor (TCR) gamma chain gene. All oral lesions were characterized by a dense and deeply infiltrative lymphoproliferation, showing epitheliotropism and massive eosinophilia. They contained atypical large lymphoid cells, which expressed T-cell markers and CD30. PCR analysis showed a monoclonal rearrangement of the TCR gamma chain gene in all lesions and, in 1 patient, the same rearrangement in the oral and cutaneous specimens. The lesions in these patients seem to be the oral counterpart of the spectrum of primary cutaneous CD30+ lymphoproliferative disorders and should be recognized as such to avoid a diagnosis of large T-cell lymphoma and possible consequent overtreatment. However, they represent only a subset among several others within the complex and heterogeneous category of disorders referred to as TEG.

Turnover of primary transcripts is a major step in the regulation of mouse H19 gene expression.

In the gene expression pathway, RNA biogenesis is a central multi-step process where both message fidelity and steady-state levels of the mature RNA have to be ascertained. An emerging question is whether RNA levels could be regulated at the precursor stage. Until recently, because it was technically very difficult to determine the level of a pre-mRNA, discrimination between changes in transcriptional activity and in pre-mRNA metabolism was extremely difficult. H19 RNA, the untranslated product of an imprinted gene, undergoes post-transcriptional regulation. Here, using a quantitative real-time RT-PCR approach, we accurately quantify its precursor RNA levels and compare these with the transcriptional activity of the gene, assessed by run-on assays. We find that the levels of H19 precursor RNA are regulated during physiological processes and this regulation appears to be related to RNA polymerase II transcription termination. Our results provide direct evidence that turnover of polymerase II primary transcripts can regulate gene expression in mammals.

Processing of 3'-extended read-through transcripts by the exosome can generate functional mRNAs.

Strains carrying rna14.1 and rna15.2 mutations are defective in pre-mRNA 3' cleavage, polyadenylation, and transcription termination. Long extended read-through transcripts generated in rna14.1 and rna15.2 strains are greatly stabilized by depletion of Rrp41p, a core component of the exosome complex or the RNA helicase Dob1p/Mtr4p. The absence of the nuclear-specific exosome component, Rrp6p, from the rna14.1 strain gave a very different phenotype. Short polyadenylated pre-mRNAs were strongly stabilized, and these were functional for translation. Production of these mRNAs was suppressed by depletion of Rrp41p, indicating that they are the products of exosome processing followed by uncoupled polyadenylation. The balance between complete degradation of 3'-unprocessed pre-mRNAs and their processing to functional mRNAs is regulated, with degradation favored on glucose media.