Human U2 snRNA Genes Exhibit a Persistently Open Transcriptional State and Promoter Disassembly at Metaphase

Abstract

In mammals, small multigene families generate spliceosomal U snRNAs that are nearly as abundant as rRNA. Using the tandemly repeated human U2 genes as a model, we show by footprinting with DNase I and permanganate that nearly all sequences between the enhancer-like distal sequence element and the initiation site are protected during interphase whereas the upstream half of the U2 snRNA coding region is exposed. We also show by chromatin immunoprecipitation that the SNAPc complex, which binds the TATA-like proximal sequence element, is removed at metaphase but remains bound under conditions that induce locus-specific metaphase fragility of the U2 genes, such as loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric p53 C terminus. We propose that the U2 snRNA promoter establishes a persistently open state to facilitate rapid reinitiation and perhaps also to bypass TFIIHdependent promoter melting; this open state would then be disassembled to allow metaphase chromatin condensation.U2 small nuclear RNA (snRNA) is the catalytic RNA component of the U2 small nuclear ribonucleoprotein particle (snRNP). Along with the U1, U4/U6, and U5 snRNPs, the U2 snRNP assembles onto eukaryotic mRNA precursors to form a spliceosome, the large multisubunit molecular machine responsible for mRNA splicing (81). The genes encoding these U snRNAs are single copy in budding yeast, where introns are rare and U snRNPs are scarce, but in mammals, where almost all mRNAs have multiple introns, the major spliceosomal U snRNAs are encoded by multigene families and the U snRNPs are nearly as abundant as rRNA. U snRNA genes have been characterized for many species, and the major transcriptional signals and trans-acting factors are well defined, especially in mammals (35,41,42). U1 to U5 are transcribed by RNA polymerase II (Pol II). The 20-bp proximal sequence element (PSE) centered at position Ϫ50 upstream from the transcription initiation site serves many of the functions of a TATA element and binds the U snRNA-specific transcription factor SNAPc (snRNA-activating protein complex; also known as PSE-binding transcription factor [PTF] and PSE-binding protein). The bipartite distal sequence element (DSE) is centered at position Ϫ235 upstream of the initiation site, serves as an enhancer, and typically binds the Sp1 and Oct-1 (or Staf/SBF) factors (90). The distance between the DSE and PSE is almost exactly one nucleosome in length and is highly conserved in metazoa; indeed, the evidence for a positioned nucleosome between the DSE and PSE is strong for U2, U6, and 7SK, suggesting that this nucleosome juxtaposes factors bound to the DSE and PSE (7, 112).In mammals, transcription apparently continues for approximately 800 bp past the U snRNA coding region (19,73). The 3Ј end of the U snRNA precursor is generated by an endonucleolytic cleavage-presumably by the Int11 component of the Integrator complex (5, 66)-just upstream from the 3Ј-end formation signal (also known as the 3Ј box) located 10 to 20 bp...

Human snRNA genes use polyadenylation factors to promote efficient transcription termination

“…How PTF affects nucleosome occupancy is currently unclear. However, our data are consistent with a previous study implicating PTF in maintaining nucleosome depletion across the U2 gene transcription unit during interphase ( 63 ). Persistent binding of PTF, in particular binding of the PTF-γ subunit, to the promoter in metaphase correlates with chromosome fragility of the RNU1/RNU2 loci.…”

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

“…S3 in the supplemental material), arguing that nucleosomal depletion of the U2 gene is not simply due to a high level of transcription. Our results argue against the presence of a stable nucleosome between the PSE and DSE of the U2 genes as previously suggested (6,45), but they are in accordance with the finding that regions within the U2 transcription unit are accessible to DNase I digestion (45). These results also indicate that transcribing pol II will encounter a high density of nucleosomes on protein-coding genes before completing a functional transcript, but not on the U2 genes, which could account for the differential requirement for P-TEFb activity for transcription elongation.…”

Sequencing the cap-snatching repertoire of H1N1 influenza provides insight into the mechanism of viral transcription initiation

“…Moreover, cap snatching of nascent transcripts explains the presence of abundant fragments corresponding to U3, U8 and U13 (Figure 4C ), despite localization of their mature forms to the nucleolus. It also explains the ∼3-fold greater abundance of leaders from U2 compared to U1, as U2 has the higher transcription rate ( 60 ) but accumulates to lower levels than U1 because of its shorter half-life ( 61 ) (Supplemental Figure S9). After accounting for nucleotides added through prime-and-realign, the lengths, nucleotide composition and identities of the host leaders were indistinguishable for the different viral mRNAs, which called into question the recently proposed influence of the viral template on the selection and utilization of the cellular transcripts ( 41 ).…”