Pumilio protects Xbp1 mRNA from regulated Ire1-dependent decay.

The unfolded protein response (UPR) maintains homeostasis of the endoplasmic reticulum (ER). Residing in the ER membrane, the UPR mediator Ire1 deploys its cytoplasmic kinase-endoribonuclease domain to activate the key UPR transcription factor Xbp1 through non-conventional splicing of Xbp1 mRNA. Ire1 also degrades diverse ER-targeted mRNAs through regulated Ire1-dependent decay (RIDD), but how it spares Xbp1 mRNA from this decay is unknown. Here, we identify binding sites for the RNA-binding protein Pumilio in the 3'UTR Drosophila Xbp1. In the developing Drosophila eye, Pumilio binds both the Xbp1unspliced and Xbp1spliced mRNAs, but only Xbp1spliced is stabilized by Pumilio. Furthermore, Pumilio displays Ire1 kinase-dependent phosphorylation during ER stress, which is required for its stabilization of Xbp1spliced. hIRE1 can phosphorylate Pumilio directly, and phosphorylated Pumilio protects Xbp1spliced mRNA against RIDD. Thus, Ire1-mediated phosphorylation enables Pumilio to shield Xbp1spliced from RIDD. These results uncover an unexpected regulatory link between an RNA-binding protein and the UPR.

Xbp1-independent Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila.

The unfolded protein response (UPR) is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Ire1 signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. Here, we show that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD), including for the fatty acid transport protein (fatp). Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. Our results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm.

AU-rich elements regulate Drosophila gene expression.

In mammals, AU-rich elements (AREs) are critical regulators of mRNA turnover. They recruit ARE-binding proteins that inhibit or stimulate rapid mRNA degradation in response to stress or developmental cues. Using a bioinformatics approach, we have identified AREs in Drosophila melanogaster 3' untranslated regions and validated their cross-species conservation in distant Drosophila genomes. We have generated a Drosophila ARE database (D-ARED) and established that about 16% of D. melanogaster genes contain the mammalian ARE signature, an AUUUA pentamer in an A/U-rich context. Using candidate ARE genes, we show that Drosophila AREs stimulate reporter mRNA decay in cultured cells and in the physiological context of the immune response in D. melanogaster. In addition, we found that the conserved ARE-binding protein Tis11 regulates temporal gene expression through ARE-mediated decay (AMD) in D. melanogaster. Our work reveals that AREs are conserved and functional cis regulators of mRNA decay in Drosophila and highlights this organism as a novel model system to unravel in vivo the contribution of AMD to various processes.

Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II).

RNA degradation is important in the post-transcriptional control of gene expression. The processing, degradation and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a 643-amino-acid enzyme that degrades single-stranded RNA from its 3'-end, releasing ribonucleoside 5'-monophosphates. RNase II was expressed both as the wild type and as a D209N mutant form. The latter was also produced as an SeMet derivative. The various protein forms were crystallized using the vapour-diffusion method. Wild-type RNase II was crystallized in two crystal forms, both of which belonged to space group P2(1). X-ray diffraction data were collected to 2.44 and 2.75 angstroms resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 angstroms, beta = 111.9 degrees and a = 119.6, b = 57.2, c = 121.2 angstroms, beta = 99.7 degrees, respectively. The RNase II D209N mutant gave crystals that belonged to space group P6(5), with unit-cell parameters a = b = 86.3, c = 279.2 angstroms, and diffracted to 2.74 angstroms. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se-atom substructure by SIRAS.

RNase R affects gene expression in stationary phase: regulation of ompA.

In nature, bacteria remain mostly in the stationary phase of the life cycle. Although mRNA is a major determinant of gene expression, little is known about mRNA decay in the stationary phase. The results presented herein demonstrate that RNase R is induced in stationary phase and is involved in the post-transcriptional regulation of ompA mRNA. This work is the first report of RNase R activity on a full length mRNA. In the absence of RNase R in a single rnr mutant, higher levels of ompA mRNA are found as a consequence of the stabilization of ompA full transcript. This effect is growth-phase-specific and not a growth-rate-dependent event. These higher levels of ompA mRNA were correlated with increases in the amounts of OmpA protein. We have also analysed the role of other factors that could affect ompA mRNA stability in stationary phase. RNase E was found to have the most important role, followed by polyadenylation. PNPase also affected the decay of the ompA transcript but RNase II did not seem to contribute much to this degradation process. The participation of RNase R in poly(A)-dependent pathways of decay in stationary phase of growth is discussed. The results show that RNase R can be a modulator of gene expression in stationary phase cells.

The role of endoribonucleases in the regulation of RNase R.

RNase R is an important exoribonuclease involved in the maturation and degradation of RNA. RNase R is co-transcribed with other genes in the same operon. In this report, we show that under physiological conditions maturation of these co-transcripts and the levels of RNase R are mainly dependent on the endoribonuclease RNase E. The presence of the full-length RNase E is necessary for the decay of intermediary products that arise from the maturation of transcripts from the rnr operon. RNase G and RNase III do not seem to have a primary role in the processing of the rnr transcripts. However, the accumulation of intermediary transcripts in an rng mutant suggests that RNase G may act in the degradation of the transcripts already cleaved by RNase E. These results demonstrated that other ribonucleases can act as an additional level of regulation in the control of the expression of RNase R.

Drosophila gene tazman, an orthologue of the yeast exosome component Rrp44p/Dis3, is differentially expressed during development.

The major 3'-5' pathway of RNA degradation in eukaryotic cells involves the exosome, which is a multi-protein complex of exoribonucleases. The exoribonucleases within this complex are highly conserved and are closely related to prokaryotic ribonucleases. We have identified and characterised the expression pattern of Drosophila tazman (taz), a component of the exosome which is closely related to Escherichia coli RNaseR and yeast Rrp44p. The tazman transcripts are differentially expressed during development, with maximum expression levels in 6-8 hr embryos. In situ hybridisation and immunolocalisation experiments show that tazman transcripts and protein are maternally derived, and are expressed ubiquitously throughout the embryo, with high levels in germ band and head structures. Differential expression of TAZ is likely to reflect changes in the activity of the 3'-5' mRNA turnover pathway which could have a major impact of the expression of target RNAs.

Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA.

In this paper we show that RNase R is a cold shock protein that is induced seven- to eightfold by cold shock and that its expression is tightly regulated by temperature. Transcriptional studies reveal that the rnr gene is co-transcribed with flanking genes as an operon induced under cold shock. The induction of RNase R levels is mainly a result of the stabilization of the rnr transcripts. The transient stability of the rnr transcripts is shown to be regulated by PNPase at the end of the acclimation phase. Studies with an rnr mutant revealed a cold-shock phenotype showing that RNase R contributes to growth at low temperatures. We have shown that RNase R can be involved in the maturation of SsrA/tmRNA, an important small stable RNA involved in protein tagging and ribosome rescue. The wide biological significance of RNase R regarding adaptation to cold shock and its involvement in RNA surveillance, protein quality control and pathogenesis is discussed.