Hertel, K. J. | Search bioRxiv

bioRxiv2024

Interdependent regulation of alternative splicing by SR and hnRNP proteins

Alternative pre-mRNA splicing is a combinatorial process involving SR and hnRNP splicing factors. These proteins can silence or enhance splicing based on their expression levels and binding positions. To better understand their combinatorial and interdependent regulation, computational analyses were performed using HepG2 and K562 cell knockdown and binding datasets from the ENCODE Project. Analyses of diMerential splicing for 6 SR proteins and 13 hnRNP knockdowns revealed statistically significant exon overlap among most RBP combinations, albeit at diMerent levels. Neither SR proteins nor hnRNPs showed strong preferences for collaborating with specific RBP classes in mediating exon inclusion. While SRSF1, hnRNPK, and hnRNPC stand out as major influencers of alternative splicing, they do so predominantly independent of other RBPs. Meanwhile, minor influencers of alternative splicing such as hnRNPAB and hnRNPA0 predominantly regulate exon inclusion in concert with other RBPs, indicating that inclusion can be mediated by both single and multiple RBPs. Interestingly, the higher the number of RBPs that regulate the inclusion of an exon, the more variable exon inclusion preferences become. Interdependently regulated exons are more modular and have diMerent physical characteristics such as reduced exon length compared to their independent counterparts. A comparison of RBP interdependence between HepG2 and K562 cells provides the framework that explains cell-type-specific alternative splicing. Our study highlights the importance of the interdependent regulation of alternative exons and identifies characteristics of interdependently regulated exons that diMer from independently regulated exons.

bioinformatics↗

bioRxiv2021

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50{micro}M) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3 and the 5 splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 {micro}M). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.

molecular biology↗