This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis–antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis–antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis–antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis–antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis–antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis–antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.

In order to study mechanisms and regulation of RNA polymerase II (RNAPII) ubiquitylation and degradation, highly purified factors were used to reconstitute RNAPII ubiquitylation in vitro. We show that arrested RNAPII elongation complexes are the preferred substrates for ubiquitylation. Accordingly, not only DNA-damage-dependent but also DNA-damage-independent transcriptional arrest results in RNAPII ubiquitylation in vivo. Def1, known to be required for damage-induced degradation of RNAPII, stimulates ubiquitylation of RNAPII only in an elongation complex. Ubiquitylation of RNAPII is dependent on its C-terminal repeat domain (CTD). Moreover, CTD phosphorylation at serine 5, a hallmark of the initiating polymerase, but not at serine 2, a hallmark of the elongating polymerase, completely inhibits ubiquitylation. In agreement with this, ubiquitylated RNAPII is hypophosphorylated at serine 5 in vivo, and mutation of the serine 5 phosphatase SSU72 inhibits RNAPII degradation. These results identify several mechanisms that confine ubiquitylation of RNAPII to the forms of the enzyme that arrest during elongation.