Identification of novel post-transcriptional features in olfactory receptor family mRNAs.

Olfactory receptor (Olfr) genes comprise the largest gene family in mice. Despite their importance in olfaction, how most Olfr mRNAs are regulated remains unexplored. Using RNA-seq analysis coupled with analysis of pre-existing databases, we found that Olfr mRNAs have several atypical features suggesting that post-transcriptional regulation impacts their expression. First, Olfr mRNAs, as a group, have dramatically higher average AU-content and lower predicted secondary structure than do control mRNAs. Second, Olfr mRNAs have a higher density of AU-rich elements (AREs) in their 3'UTR and upstream open reading frames (uORFs) in their 5 UTR than do control mRNAs. Third, Olfr mRNAs have shorter 3' UTR regions and with fewer predicted miRNA-binding sites. All of these novel properties correlated with higher Olfr expression. We also identified striking differences in the post-transcriptional features of the mRNAs from the two major classes of Olfr genes, a finding consistent with their independent evolutionary origin. Together, our results suggest that the Olfr gene family has encountered unusual selective forces in neural cells that have driven them to acquire unique post-transcriptional regulatory features. In support of this possibility, we found that while Olfr mRNAs are degraded by a deadenylation-dependent mechanism, they are largely protected from this decay in neural lineage cells

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages

A microRNA cluster in the Fragile-X region expressed during spermatogenesis targets FMR1.

Testis-expressed X-linked genes typically evolve rapidly. Here, we report on a testis-expressed X-linked microRNA (miRNA) cluster that despite rapid alterations in sequence has retained its position in the Fragile-X region of the X chromosome in placental mammals. Surprisingly, the miRNAs encoded by this cluster (Fx-mir) have a predilection for targeting the immediately adjacent gene, Fmr1, an unexpected finding given that miRNAs usually act in trans, not in cis Robust repression of Fmr1 is conferred by combinations of Fx-mir miRNAs induced in Sertoli cells (SCs) during postnatal development when they terminate proliferation. Physiological significance is suggested by the finding that FMRP, the protein product of Fmr1, is downregulated when Fx-mir miRNAs are induced, and that FMRP loss causes SC hyperproliferation and spermatogenic defects. Fx-mir miRNAs not only regulate the expression of FMRP, but also regulate the expression of eIF4E and CYFIP1, which together with FMRP form a translational regulatory complex. Our results support a model in which Fx-mir family members act cooperatively to regulate the translation of batteries of mRNAs in a developmentally regulated manner in SCs

Biological role of phosphate starvation induced genes in the maintenance of phosphate homeostasis in Arabidopsis thaliana

Phosphate (Pi) plays a central role in several metabolic processes and is an indispensable building block for the biosynthesis of nucleic acids and phospholipids. As a response to Pi deficiency the plant undergoes an array of morphophysiological, biochemical and molecular adaptations. In order to better understand these adaptive mechanisms, three candidate genes selected from a microarray analysis were characterized for their role during Pi stress. These three genes — an organic phosphate transporter, a transcription factor and a RING E3 ligase are induced during Pi starvation. Both forward and reverse genetics approach were used to define their biological function. Glycerol-3-phosphate permease (G3Pp) is a Pi/anion antiporter represented by a gene family of five members in Arabidopsis. The spatio-temporal expression pattern of members of this gene family during Pi stress was characterized by transcript analysis and generation of transgenic plants carrying promoter-GUS fusion constructs. All the five members were induced by Pi starvation but varied in their tissue specific expression. Also, the distinct expression of the G3Pps in the early stages of germination suggests a role in nutrient mobilization during this metabolically active stage. Characterization of a knock-down mutant for one of these transporters revealed a developmentally regulated functional compensation by other members of the gene family Transcription factors like AtERF070 form a part of regulatory networks as they can modulate the expression of target genes. AtERF070 belongs to the plant specific AP2 domain containing Ethylene Response Factor (ERF) family. RNAi mediated suppression of AtERF070 resulted in an increase in the number of lateral roots and root hairs which could have contributed to higher Pi content in the transgenic plants. Over-expression of this transcription factor caused a gibberellic acid (GA)-deficient phenotype. They exhibited delayed senescence, late flowering, increased accumulation of anthocyanin and reduction in the number of lateral roots which could be partially rescued by the exogenous application of the phytohormone GA. The expression of several GA biosynthetic genes was reduced, further corroborating the possibility of altered phytohormone levels in the over-expressor. Several Pi-starvation induced (PSI) genes especially microRNAs and RNAses were found to be upregulated by the over-expression of AtERF070 . These results suggest the involvement of AtERF070 in maintenance of Pi homeostasis and GA-mediated growth modulation during Pi starvation. The role of ATL8 , a RING E3 ligase induced during Pi deficiency was also investigated as a part of this study. Modulation of this gene by Pi starvation indicates a plausible post translational regulation of PSI responses. A T-DNA insertion mutant of ATL8 exhibited a reduction in lateral root number and a subsequent decrease in accumulation of Pi. In contrast, over-expression led to an abscissic acid resistant phenotype and more number of lateral roots leading to increased accumulation of Pi. Based on the above results we can conclude that modulation of the root system by PSI genes is an important biological adaptation during Pi deficiency. The modulation of plant growth is mediated by phytohormones like GA, cytokinin, auxin and involves genes like AtERF070 and ATL8 . These observations point towards a complex transcriptional and post translational regulation of the adaptations during Pi starvation

Identification of novel post-transcriptional features in olfactory receptor family mRNAs.

Olfactory receptor (Olfr) genes comprise the largest gene family in mice. Despite their importance in olfaction, how most Olfr mRNAs are regulated remains unexplored. Using RNA-seq analysis coupled with analysis of pre-existing databases, we found that Olfr mRNAs have several atypical features suggesting that post-transcriptional regulation impacts their expression. First, Olfr mRNAs, as a group, have dramatically higher average AU-content and lower predicted secondary structure than do control mRNAs. Second, Olfr mRNAs have a higher density of AU-rich elements (AREs) in their 3'UTR and upstream open reading frames (uORFs) in their 5 UTR than do control mRNAs. Third, Olfr mRNAs have shorter 3' UTR regions and with fewer predicted miRNA-binding sites. All of these novel properties correlated with higher Olfr expression. We also identified striking differences in the post-transcriptional features of the mRNAs from the two major classes of Olfr genes, a finding consistent with their independent evolutionary origin. Together, our results suggest that the Olfr gene family has encountered unusual selective forces in neural cells that have driven them to acquire unique post-transcriptional regulatory features. In support of this possibility, we found that while Olfr mRNAs are degraded by a deadenylation-dependent mechanism, they are largely protected from this decay in neural lineage cells

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages