SummaryThe transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal ∼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.

SummaryRECQL5 is the sole member of the RECQ family of helicases associated with RNA polymerase II (RNAPII). We now show that RECQL5 is a general elongation factor that is important for preserving genome stability during transcription. Depletion or overexpression of RECQL5 results in corresponding shifts in the genome-wide RNAPII density profile. Elongation is particularly affected, with RECQL5 depletion causing a striking increase in the average rate, concurrent with increased stalling, pausing, arrest, and/or backtracking (transcription stress). RECQL5 therefore controls the movement of RNAPII across genes. Loss of RECQL5 also results in the loss or gain of genomic regions, with the breakpoints of lost regions located in genes and common fragile sites. The chromosomal breakpoints overlap with areas of elevated transcription stress, suggesting that RECQL5 suppresses such stress and its detrimental effects, and thereby prevents genome instability in the transcribed region of genes.

Synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is regulated by the mTOR pathway in mammalian cells. The mTOR kinase localizes to tRNA and 5S rRNA genes, providing an opportunity for direct control. Its presence at these sites can be explained by interaction with TFIIIC, a DNA-binding factor that recognizes the promoters of these genes. TFIIIC contains a TOR signaling motif that facilitates its association with mTOR. Maf1, a repressor that binds and inhibits pol III, is phosphorylated in a mTOR-dependent manner both in vitro and in vivo at serine 75, a site that contributes to its function as a transcriptional inhibitor. Proximity ligation assays confirm the interaction of mTOR with Maf1 and TFIIIC in nuclei. In contrast to Maf1 regulation in yeast, no evidence is found for nuclear export of Maf1 in response to mTOR signaling in HeLa cells. We conclude that mTOR associates with TFIIIC, is recruited to pol III–transcribed genes, and relieves their repression by Maf1.

Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis.

We have previously identified poly(A)-binding protein 1 (PABP1) as a ligand for paxillin and shown that the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling. By targeting the paxillin-binding subdomain sequences in PABP1, we have generated mutants of PABP1 that do not bind to cellular paxillin. Here we report that paxillin association is necessary for efficient nuclear export of PABP1 and that RNA interference of paxillin drives the nuclear accumulation of PABP1. Furthermore, ablation of paxillin-PABP1 association impeded a number of indices of cell motility including spreading on fibronectin, cell migration on twodimensional matrices, and transmigration in Boyden chambers. These data indicate that PABP1 must associate with paxillin in order to be efficiently transported from the nucleus to the cytoplasm and that this event is necessary for cells to remodel their focal adhesions during cell migration.The leading edge or lamellipodium of migrating cells is the point at which integrins engage the extracellular matrix, an interaction that leads to integrin clustering into an array of small multiprotein focal complexes (31) containing an array of structural and signaling proteins that act to establish and maintain the polarity of the migrating cell. The 68-kDa protein paxillin is an abundant component of focal complexes at the leading edge of migrating cells. In addition to binding directly to integrin cytodomains, paxillin contains numerous proteinbinding modules that interact with a variety of structural and signaling proteins and is therefore classified as a molecular adaptor or scaffold protein (23). Paxillin has an N-terminal region with five leucine-rich motifs, termed LD domains, and a C-terminal portion with four tandem LIM domains. The LIM domains contain information for targeting paxillin to focal complexes and can bind directly to tubulin (9). LD domains are protein-protein interaction motifs with the consensus sequence LDXXLLXXL, and these mediate the interaction of paxillin with a number of proteins that regulate cell migration. LD domains seem to display some degree of selectivity with respect to the ligands they recruit; LD1 binds integrin-linked kinase and the actin-binding proteins vinculin and actopaxin, while LD2 associates with focal adhesion kinase (FAK) and the ARF-GAP protein, p95PKL (23). Based on mutational analysis, Turner and coworkers (23) have identified the regions of proteins that associate with the various LD domains and termed them paxillin-binding subdomains or PBSs.Using a proteomic approach, we have recently identified an association between paxillin and the mRNA-binding protein, PABP1. Moreover, the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling and is localized to sites of translation in the perinuclear endoplasmic reticulum and at the leading edge of migrating cells (29). PABP1 consists of an N-terminal portion that contains four tandem RNA-binding motifs (RRM) and a C-terminal region with homology to an ubiquitin E3 ligase, called HYD (Fig. 1A) (12). ...