Embryonic development of
Coupling of microRNA-directed phased small interfering RNA generation from long noncoding genes with alternative splicing and alternative polyadenylation in small RNA-mediated gene silencing
More Like this
-
more » « less
Abstract Austrofundulus limnaeus can occur along two phenotypic trajectories that are physiologically and biochemically distinct. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately non-diapausing embryos and older females produce mostly diapausing embryos. Embryonic incubation temperature can override this pattern and alter trajectory. We hypothesized that temperature-induced phenotypic plasticity may be regulated by post-transcriptional modification via noncoding RNAs. As a first step to exploring this possibility, RNA-seq was used to generate transcriptomic profiles of small noncoding RNAs in embryos developing along the two alternative trajectories. We find distinct profiles of mature sequences belonging to the miR-10 family expressed in increasing abundance during development and mature sequences of miR-430 that follow the opposite pattern. Furthermore, miR-430 sequences are enriched in escape trajectory embryos. MiR-430 family members are known to target maternally provisioned mRNAs in zebrafish and may operate similarly inA. limnaeus in the context of normal development, and also by targeting trajectory-specific mRNAs. This expression pattern and function for miR-430 presents a potentially novel model for maternal-embryonic conflict in gene regulation that provides the embryo the ability to override maternal programming in the face of altered environmental conditions. -
Splicing is highly regulated and is modulated by numerous factors. Quantitative predictions for how a mutation will affect precursor mRNA (pre-mRNA) structure and downstream function are particularly challenging. Here, we use a novel chemical probing strategy to visualize endogenous precursor and mature MAPT mRNA structures in cells. We used these data to estimate Boltzmann suboptimal structural ensembles, which were then analyzed to predict consequences of mutations on pre-mRNA structure. Further analysis of recent cryo-EM structures of the spliceosome at different stages of the splicing cycle revealed that the footprint of the B act complex with pre-mRNA best predicted alternative splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene, achieving 74% accuracy. We further developed a β-regression weighting framework that incorporates splice site strength, RNA structure, and exonic/intronic splicing regulatory elements capable of predicting, with 90% accuracy, the effects of 47 known and 6 newly discovered mutations on inclusion of exon 10 of MAPT . This combined experimental and computational framework represents a path forward for accurate prediction of splicing-related disease-causing variants.more » « less
-
Alternate isoforms are important contributors to phenotypic diversity across eukaryotes. Although short-read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full-length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we use Pacific Biosciences (PacBio) long-read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five organs in threespine stickleback fish ( Gasterosteus aculeatus ), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly in which gene annotations are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq-predicted transcription start sites were more accurate and verified through ATAC-seq. We also detected many alternative splicing events between sexes and across organs. We found a substantial number of genes in both somatic and gonadal samples that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.more » « less
-
(1) Background: A simplistic understanding of the central dogma falls short in correlating the number of genes in the genome to the number of proteins in the proteome. Post-transcriptional alternative splicing contributes to the complexity of the proteome and is critical in understanding gene expression. mRNA-sequencing (RNA-seq) has been widely used to study the transcriptome and provides opportunity to detect alternative splicing events among different biological conditions. Despite the popularity of studying transcriptome variants with RNA-seq, few efficient and user-friendly bioinformatics tools have been developed for the genome-wide detection and visualization of alternative splicing events. (2) Results: We propose AS-Quant, (Alternative Splicing Quantitation), a robust program to identify alternative splicing events from RNA-seq data. We then extended AS-Quant to visualize the splicing events with short-read coverage plots along with complete gene annotation. The tool works in three major steps: (i) calculate the read coverage of the potential spliced exons and the corresponding gene; (ii) categorize the events into five different categories according to the annotation, and assess the significance of the events between two biological conditions; (iii) generate the short reads coverage plot for user specified splicing events. Our extensive experiments on simulated and real datasets demonstrate that AS-Quant outperforms the other three widely used baselines, SUPPA2, rMATS, and diffSplice for detecting alternative splicing events. Moreover, the significant alternative splicing events identified by AS-Quant between two biological contexts were validated by RT-PCR experiment. (3) Availability: AS-Quant is implemented in Python 3.0. Source code and a comprehensive user’s manual are freely available online.more » « less
