Newbury (2015) The 3'-5' exoribonuclease Dis3 regulates the expression of specific microRNAs in Drosophila wing imaginal discs, RNA Biology, 12:7, 728-741, DOI: 10.1080/15476286.2015 Keywords: Dis3/Taz, Drosophila development, exoribonuclease, imaginal discs, miRNAs, RNA stability, RNA degradation Dis3 is a highly conserved exoribonuclease which degrades RNAs in the 3'-5' direction. Mutations in Dis3 are associated with a number of human cancers including multiple myeloma and acute myeloid leukemia. In this work, we have assessed the effect of a Dis3 knockdown on Drosophila imaginal disc development and on expression of mature microRNAs. We find that Dis3 knockdown severely disrupts the development of wing imaginal discs in that the flies have a "no wing" phenotype. Use of RNA-seq to quantify the effect of Dis3 knockdown on microRNA expression shows that Dis3 normally regulates a small subset of microRNAs, with only 11 (10.1%) increasing in level 2-fold and 6 (5.5%) decreasing in level 2-fold. Of these microRNAs, miR-252-5p is increased 2.1-fold in Dis3-depleted cells compared to controls while the level of the miR-252 precursor is unchanged, suggesting that Dis3 can act in the cytoplasm to specifically degrade this mature miRNA. Furthermore, our experiments suggest that Dis3 normally interacts with the exosomal subunit Rrp40 in the cytoplasm to target miR-252-5p for degradation during normal wing development. Another microRNA, miR-982-5p, is expressed at lower levels in Dis3 knockdown cells, while the miR-982 precursor remains unchanged, indicating that Dis3 is involved in its processing. Our study therefore reveals an unexpected specificity for this ribonuclease toward microRNA regulation, which is likely to be conserved in other eukaryotes and may be relevant to understanding its role in human disease.

RNA molecules are subjected to post-transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can occur within the RNA molecule or at its 5 0 -or 3 0 -extremities, and are essential for gene regulation and proper function of the RNA. One major type of modification is the 3 0 -end addition of nontemplated nucleotides. Polyadenylation is the most well studied type of 3 0 -RNA modification, both in eukaryotes and prokaryotes. The importance of 3 0 -oligouridylation has recently gained attention through the discovery of several types of uridylated-RNAs, by the existence of enzymes that specifically add poly(U) tails and others that preferentially degrade these tails. Namely, Dis3L2 is a 3 0 -5 0 exoribonuclease from the RNase II/RNB family that has been shown to act preferentially on oligo(U)-tailed transcripts. Our understanding of this process is still at the beginning, but it is already known to interfere in the regulation of diverse RNA species in most eukaryotes. Now that we are aware of the prevalence of RNA uridylation and the techniques available to globally evaluate the 3 0 -terminome, we can expect to make rapid progress in determining the extent of terminal oligouridylation in different RNA populations and unravel its impact on RNA decay mechanisms. Here, we sum up what is known about 3 0 -RNA modification in the different cellular compartments of eukaryotic cells, the conserved enzymes that perform this 3 0 -end modification and the effectors that are selectively activated by this process.

RNA half-lives are frequently perceived as depending on too many variables, and transcript stability is generally missed as a checkpoint amenable to manipulation in synthetic designs. In this work, the contribution of mRNA stability to heterologous protein production levels in E. coli has been inspected. To this end, we capitalized on the wealth of information available on intrinsic mRNA stability determinants, four of which were formatted as portable modules consisting of 5'-untranslated regions (UTRs). The cognate DNA sequences were then assembled in a genetic frame in which mRNA stability endowed by the UTRs was the only variable to run expression of sfGFP. Reporter output and Northern blot-based measurements of absolute mRNA half-lives revealed that such UTRs were found to keep intact their ability to modulate transcript stability when excised from their natural context and placed as the upstream region of the reporter gene. By keeping transcription fixed and combining different UTRs with a constant ribosomal binding site, we showed that mRNA decay can be made the limiting constituent of the overall gene expression flow. Moreover, the data indicated that manipulating mRNA stability had little effect on expression noise in the corresponding population. Finally, augmented heterologous expression brought about by mRNA stability did not make cells more vulnerable to resource-consuming stresses. The tangible result of this work was a collection of well-characterized mRNA-stabilizing sequences that can be composed along with other expression signals in any construct following the assembly rules of the Standard European Vector Architecture (SEVA) format.