DOI: 10.5061/dryad.k0p2ngfj3
title: Transcriptome-wide alternative mRNA splicing analysis reveals post-transcriptional regulation of neuronal differentiation

description: Alternative splicing (AS) plays an important role in neuronal development,
 function, and disease. Efforts to analyze the transcriptome of AS neurons
 on a wide scale are currently limited. We characterized the
 transcriptome-wide AS changes in SH-SY5Y neuronal differentiation model,
 which is widely used to study neuronal function and disorders. Our
 analysis revealed global changes in five AS programs that drive neuronal
 differentiation. Motif analysis revealed the contribution of RNA binding
 proteins (RBPs) to the regulation of AS during neuronal development. We
 concentrated on the primary alternative splicing program that occurs
 during differentiation, specifically on events involving exon skipping
 (SE). Motif analysis revealed motifs for Polypyrimidine Tract Binding
 Protein 1 (PTB) and ELAV Like RNA Binding Protein 1 (HuR/ELAVL1) to be the
 top enriched in SE events, and their protein levels were downregulated
 after differentiation. shRNA Knockdown of either PTB and HuR was
 associated with enhanced neuronal differentiation and transcriptome-wide
 exon skipping events that drive the process of differentiation. At the
 level of gene expression, we observed only modest changes, indicating
 predominant post-transcriptional effects of PTB and HuR. We also observed
 that both RBPs altered cellular responses to oxidative stress, in line
 with the differentiated phenotype observed after either gene knockdown.
 Our work characterizes the AS changes in a widely used and important model
 of neuronal development and neuroscience research and reveals intricate
 post-transcriptional regulation of neuronal differentiation.
description: # Transcriptome-wide alternative mRNA splicing analysis reveals
 post-transcriptional regulation of neuronal differentiation
 [https://doi.org/10.5061/dryad.k0p2ngfj3](https://doi.org/10.5061/dryad.k0p2ngfj3) ## Description of the data and file structure Supplementary tables containing local splice variants (LSV) events generated using the rMATs pipeline. The data include 5 LSV subtypes analyzed in Differentiated vs undifferentiated SH-SY5Y cells as well as after PTB or HuR Knockdown experiments. For detailed bioinformatics analysis, please refere to the original article. ### Files and variables #### File: Supplementary\_tables.xlsx **Description:** Supplementary tables containing LSV data from RNA-seq experiments. ##### Variables Supplementary table 1: exon skipping events after neural differentiation (Differentiated vs undifferentiated cells) Supplementary table 2: mutually exclusive exon events after neural differentiation (Differentiated vs undifferentiated cells) Supplementary table 3: retained intron events after neural differentiation (Differentiated vs undifferentiated cells) Supplementary table 4: alternative 5’ splice site events after neural differentiation (Differentiated vs undifferentiated cells) Supplementary table 5: alternative 3’ splice site events after neural differentiation (Differentiated vs undifferentiated cells) Supplementary table 6: exon skipping events after PTB KD (KD vs Mock) Supplementary table 7: mutually exclusive exon events after PTB KD (KD vs Mock) Supplementary table 8: retained intron events after PTB KD (KD vs Mock) Supplementary table 9: alternative 5’ splice site events after PTB KD (KD vs Mock) Supplementary table 10: alternative 3’ splice site events after PTB KD (KD vs Mock) Supplementary table 11: exon skipping events after HuR KD (KD vs Mock) Supplementary table 12: mutually exclusive exon events after HuR KD (KD vs Mock) Supplementary table 13: retained intron events after HuR KD (KD vs Mock) Supplementary table 14: alternative 5’ splice site events after HuR KD (KD vs Mock) Supplementary table 15: alternative 3’ splice site events after HuR KD (KD vs Mock) ## Access information Data was derived from the following sources: * RNA sequencing data of PTB and HuR KD was deposited in the sequence read archive (PRJNA1001516). The RNA-seq data of differentiated neurons was retrieved from previous publication (Zhou et al, J Cell Phys, 2024) and is available through the sequence read archive (PRJNA779467). * Bioinformatics analysis of RNA-seq data: Quality control for Raw Fastq was performed using FastQC. Raw reads were then trimmed with Trimmomatic and adaptor sequences and low-quality read removed. Reads were aligned to the human reference genome hg38 (GRCh38.p13) using the splice aware aligner HISAT2. Mapped reads BAM file were then counted to gene features by FeatureCounts with standard settings. Cutoff for significant gene expression was FDR &lt; 0.05 and |Log2 FC| ≥ 1. Local splice variants (LSV) analysis was conducted using rMATs and motif analysis of RBPs was conducted using rMAPs2. Cutoff value for statistically significant splice event was FDR &lt; 0.05 and |ΔΨ| ≥ 0.2. Pre ranked gene set enrichment analysis was conducted using eVITTA toolbox. Overrepresentation analysis (ORA) was conducted using eVITTA or Metascape. * For datasheet structure and explanation, please refer to rMATs documentations: https://github.com/Xinglab/rmats-turbo/blob/v4.1.1/README.md