Checkpoint kinase interaction with DNA polymerase alpha regulates replication progression during stress.

Background: In eukaryotes, replication stress activates a checkpoint response, which facilitates genome duplication by stabilising the replisome. How the checkpoint kinases regulate the replisome remains poorly understood. The aim of this study is to identify new targets of checkpoint kinases within the replisome during replication stress. Methods: Here we use an unbiased biotin proximity-ligation approach in Saccharomyces cerevisiae to identify new interactors and substrates of the checkpoint kinase Rad53 in vivo. Results: From this screen, we identified the replication initiation factor Sld7 as a Rad53 substrate, and Pol1, the catalytic subunit of polymerase a, as a Rad53-interactor. We showed that CDK phosphorylation of Pol1 mediates its interaction with Rad53. Combined with other interactions between Rad53 and the replisome, this Rad53-Pol1 interaction is important for viability and replisome progression during replication stress. Conclusions: Together, we explain how the interactions of Rad53 with the replisome are controlled by both replication stress and the cell cycle, and why these interactions might be important for coordinating the stabilisation of both the leading and lagging strand machineries.

Noncanonical RNAs from transcripts of the Drosophila muscleblind gene.

It has become increasingly evident that eukaryotic cells produce RNA molecules from coding genes with constitutions other than those of typically spliced mRNA transcripts. Here we describe new cDNAs from the Drosophila melanogaster muscleblind (mbl) locus that identify two such atypical RNA molecules: RNAs containing an incomplete exon 2 tandem repetition (mblE2E2') or having exons with a different order compared to the corresponding genomic DNA (mblE2E3'E2'; exon scrambling). The existence of exon duplications and rearrangements in the genomic locus that might explain such cDNAs was ruled out by genomic Southern blotting and in silico analysis of the Drosophila genome sequence. The incomplete exon 2 tandem repetition was confirmed by sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) products, rapid amplification of cDNA ends, and detection of a band consistent with cDNA sizes in total RNA northern blots. RT-PCRs with exon-specific primers downstream of exon 2 were unable to amplify products other than those expected from canonical mbl isoforms, thus indicating that no other exons were efficiently spliced downstream of exon 2. Moreover, mblE2E2' transcripts seem to be poorly polyadenylated, if at all, and behave aberrantly in a polyacrylamide gel electrophoresis (PAGE) mobility assay. Taken together, lack of polyadenylation, lack of downstream splicing events, small size of mblE2E2', and PAGE behavior all suggest that these noncanonical transcripts may be circular RNAs. The functional implications for these noncanonical transcripts are unclear. A developmental expression profile of mblE2E2' revealed an almost constant expression except during early embryogenesis and early adulthood. The protein putatively encoded is unlikely to be functional because an in-frame stop codon occurs almost immediately after the splice site. Such noncanonical transcripts have previously been observed in vertebrates, and these data provide the first experimental evidence for similar phenomena in invertebrates.

Myotonic dystrophy associated expanded CUG repeat muscleblind positive ribonuclear foci are not toxic to Drosophila.

Myotonic dystrophy type 1 is an autosomal dominant disorder associated with the expansion of a CTG repeat in the 3' untranslated region (UTR) of the DMPK gene. Recent data suggest that pathogenesis is predominantly mediated by a gain of function of the mutant transcript. In patients, these expanded CUG repeat-containing transcripts are sequestered into ribonuclear foci that also contain the muscleblind-like proteins. To provide further insights into muscleblind function and the pathogenesis of myotonic dystrophy, we generated Drosophila incorporating CTG repeats in the 3'-UTR of a reporter gene. As in patients, expanded CUG repeats form discrete ribonuclear foci in Drosophila muscle cells that co-localize with muscleblind. Unexpectedly, however, foci are not observed in all cell types and muscleblind is neither necessary nor sufficient for their formation. The foci are dynamic transient structures with short half-lifes that do not co-localize with the proteasome, suggesting they are unlikely to contain mis-folded proteins. However, they do co-localize with non-A, the human orthologs of which are implicated in both RNA splicing and attachment of dsRNA to the nuclear matrix. Muscleblind is also revealed as having a previously unrecognized role in stabilizing CUG transcripts. Most interestingly, Drosophila expressing (CUG)162 repeats has no detectable pathological phenotype suggesting that in contrast to expanded polyglutamine-containing proteins, neither the expanded CUG repeat RNA nor the ribonuclear foci are directly toxic.