Regulation of mRNA decay by Pumilio in Drosophila melanogaster

During early embryogenesis in all animals, development is driven by maternally loaded mRNAs synthesized in the female parent during oogenesis. These maternally synthesized mRNAs are regulated by post-transcriptional mechanisms, including mRNA localization, degradation and translational activation and repression. Many of these processes rely on RNA-binding proteins to initially recognize and bind specific mRNAs and then, subsequently, recruit the appropriate post-transcriptional machinery. Eventually the products encoded by the zygotic genome regulate embryo development. My thesis focuses on an RNA-binding protein called Pumilio and its role in regulating the degradation of maternal mRNAs in Drosophila melanogaster embryos. Pumilio protein and pumilio mRNA are both maternally loaded and deficiencies in pumilio are maternal-effect lethal. While homologs of Pumilio had been implicated in mediating mRNA degradation, the global role of Pumilio itself had been unclear, with contradictory results from embryos and whole flies with a conditional pumilio mutant genotype. I first produced transgenic flies expressing a tandem affinity tagged Pumilio transgene. Then, by pulling down tagged Pumilio followed by next-generation sequencing of co-purifying RNAs, I found that the Pumilio protein associates with ~200 mRNAs in early embryos. Using a null pumilio allele combination, I found that Pumilio regulates the decay of a set of mRNAs in a manner that, in part, appears to be dependent on zygotically encoded factors. I observed that many of the mRNAs whose stability is affected in pumilio mutant embryos are amongst those physically associated with Pumilio protein in early embryos and validated my findings using reporter transgenes. My data show that Pumilio is a regulator of maternal transcript degradation in D. melanogaster.Ph

Brain tumor is a sequence-specific RNA-binding protein that directs maternal mRNA clearance during the Drosophila maternal-to-zygotic transition

Abstract Background Brain tumor (BRAT) is a Drosophila member of the TRIM-NHL protein family. This family is conserved among metazoans and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. Results Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. The regulatory status of BRAT-associated mRNAs suggests a prominent role for BRAT in post-transcriptional regulation, including a previously unidentified role in transcript degradation. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to-zygotic transition. Conclusions Our results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM

Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations

Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

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