The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions

Ptbp2 Controls an Alternative Splicing Network Required for Cell Communication during Spermatogenesis

Summary: Alternative splicing has essential roles in development. Remarkably, spermatogenic cells express more alternatively spliced RNAs compared to most whole tissues; however, regulation of these RNAs remains unclear. Here, we characterize the alternative splicing landscape during spermatogenesis and reveal an essential function for the RNA-binding protein Ptbp2 in this highly regulated developmental program. We found that Ptbp2 controls a network of genes involved in cell adhesion, migration, and polarity, suggesting that splicing regulation by Ptbp2 is critical for germ cell communication with Sertoli cells (multifunctional somatic cells necessary for spermatogenesis). Indeed, Ptbp2 ablation in germ cells resulted in disorganization of the filamentous actin (F-actin) cytoskeleton in Sertoli cells, indicating that alternative splicing regulation is necessary for cellular crosstalk during germ cell development. Collectively, the data delineate an alternative splicing regulatory network essential for spermatogenesis, the splicing factor that controls it, and its biological importance in germ-Sertoli communication. : Hannigan et al. characterize the alternative splicing landscape during spermatogenesis and reveal an essential role for Ptbp2 in this temporal regulation. Moreover, splicing regulation by Ptbp2 is required for proper regulation of trafficking and cell polarity genes and cell communication during germ cell development. Keywords: post-transcriptional regulation, spermatogenesis, cell-cell communication, alternative splicing, RNA network

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response.

Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of newly synthesized RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation

Argonaute High-Throughput Sequencing of RNAs Isolated by Cross-Linking Immunoprecipitation Reveals a Snapshot of miRNA Gene Regulation in the Mammalian Retina

Mounting evidence points to roles for miRNA gene regulation in promoting development, function, and cell survival in the mammalian retina. However, little is known regarding which retinal genes are targets of miRNAs. Here, we employed a systematic, nonbiased, biochemical approach to identify targets of miRNA gene regulation in the bovine retina, a common model species for vision research. Using Argonaute high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation analysis, we identified 348 high-confidence miRNA target sites within 261 genes. This list was enriched in rod and cone photoreceptor genes and included 28 retinal disease genes, providing further evidence of a role of miRNAs in the pathology of blinding diseases

Argonaute High-Throughput Sequencing of RNAs Isolated by Cross-Linking Immunoprecipitation Reveals a Snapshot of miRNA Gene Regulation in the Mammalian Retina

Mounting evidence points to roles for miRNA gene regulation in promoting development, function, and cell survival in the mammalian retina. However, little is known regarding which retinal genes are targets of miRNAs. Here, we employed a systematic, nonbiased, biochemical approach to identify targets of miRNA gene regulation in the bovine retina, a common model species for vision research. Using Argonaute high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation analysis, we identified 348 high-confidence miRNA target sites within 261 genes. This list was enriched in rod and cone photoreceptor genes and included 28 retinal disease genes, providing further evidence of a role of miRNAs in the pathology of blinding diseases

The helicase Ded1p controls use of near-cognate translation initiation codons in 5′ UTRs

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation 1 . Mutations in DDX3 are linked to tumorigenesis 2-4 and intellectual disability 5, and the enzyme is targeted by a range of viruses 6 . How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5′ untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5′ untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5′ untranslated regions

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions

Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions

Argonaute High-Throughput Sequencing of RNAs Isolated by Cross-Linking Immunoprecipitation Reveals a Snapshot of miRNA Gene Regulation in the Mammalian Retina

[Image: see text] Mounting evidence points to roles for miRNA gene regulation in promoting development, function, and cell survival in the mammalian retina. However, little is known regarding which retinal genes are targets of miRNAs. Here, we employed a systematic, nonbiased, biochemical approach to identify targets of miRNA gene regulation in the bovine retina, a common model species for vision research. Using Argonaute high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation analysis, we identified 348 high-confidence miRNA target sites within 261 genes. This list was enriched in rod and cone photoreceptor genes and included 28 retinal disease genes, providing further evidence of a role of miRNAs in the pathology of blinding diseases