Transcriptome analysis of developing lens reveals abundance of novel transcripts and extensive splicing alterations

Abstract

Lens development involves a complex and highly orchestrated regulatory program. Here, we investigate the transcriptomic alterations and splicing events during mouse lens formation using RNA-seq data from multiple developmental stages, and construct a molecular portrait of known and novel transcripts. We show that the extent of novelty of expressed transcripts decreases significantly in post-natal lens compared to embryonic stages. Characterization of novel transcripts into partially novel transcripts (PNTs) and completely novel transcripts (CNTs) (novelty score ≥ 70%) revealed that the PNTs are both highly conserved across vertebrates and highly expressed across multiple stages. Functional analysis of PNTs revealed their widespread role in lens developmental processes while hundreds of CNTs were found to be widely expressed and predicted to encode for proteins. We verified the expression of four CNTs across stages. Examination of splice isoforms revealed skipped exon and retained intron to be the most abundant alternative splicing events during lens development. We validated by RT-PCR and Sanger sequencing, the predicted splice isoforms of several genes Banf1, Cdk4, Cryaa, Eif4g2, Pax6, and Rbm5. Finally, we present a splicing browser Eye Splicer (http://www.iupui.edu/~sysbio/eye-splicer/), to facilitate exploration of developmentally altered splicing events and to improve understanding of post-transcriptional regulatory networks during mouse lens development.

Long non-coding RNA expression levels modulate cell-type specific splicing patterns by altering their interaction landscape with RNA-binding proteins

“…Reports on the interactions between lncRNAs in unique human cell lines are becoming more prevalent as well curated databases are emerging to improve the quality of lncRNA annotation [6,47]. As more lncRNA knockdown procedures and RBP binding data are released to the public, pipelines like the one conducted in this study can be utilized to investigate the mechanisms of lncRNAs in alternative splicing.…”

Long Non-Coding RNA Expression Levels Modulate Cell-Type Specific Splicing Patterns by Altering Their Interaction Landscape with RNA-Binding Proteins

“…However, the sponge model was not able to represent all lncRNAs which played a significant role in splicing. LncRNAs, like RP5-1148A21.3, which affected splicing very significantly and had few Reports on the interactions between lncRNAs in unique human cell lines are becoming more prevalent as well curated databases are emerging to improve the quality of lncRNA annotation [6,47].…”

Role of SARS-CoV-2 in Altering the RNA-Binding Protein and miRNA-Directed Post-Transcriptional Regulatory Networks in Humans

“…The quality of the sequence reads was ensured using the FASTX-Toolkit ( ), with a minimum of Phred quality score 20 for each sample. We processed the raw sequencing reads using the in-house NGS data processing pipeline, as described previously [ 99 , 100 ]. Briefly, we used Hierarchical Indexing for the Spliced Alignment of Transcripts (HISAT) [ 101 ] for aligning the short reads from the RNA-seq experiments onto the human reference genome (hg38).…”

“…It also uses a GTF file (gene transfer file format), downloaded from Ensembl (version 97) [ 107 ] for the existing annotation of exons. Briefly, rMATS enabled the analysis of the inclusion/exclusion of target exons/introns, contributing to different types of alternative splicing events, namely skipped exon (SE), alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE), and retained intron (RI), between pairs of conditions and provided the difference in the level of inclusion of an exon denoted by the Percentage Splicing Index ( ) (as described previously [ 99 ]). Genes exhibiting alternatively spliced events detected below the 5% FDR threshold were documented in Supplementary Table S2 .…”