Randazza, A. | Search bioRxiv

bioRxiv2026

Neural microexons contain lengthened sequence and extended RNA structure between the branchpoint and splice site motif

Microexons are short exons that are highly conserved in vertebrates and are essential for neurodevelopment. Their small size poses a challenge for regulatory protein binding and exon-definition splice site recognition, which typically relies on standard length exons. Here, we determine the sequence and RNA structural features of neural microexons in humans and in the chick developmental model organism. We demonstrate that a subset of neural microexons undergoes dynamic, stage-specific regulation during chick embryonic brain development that correlates with expression of known microexon regulators, SRRM4 and NOVA1. Using experimental RNA structure-probing on a subset of neural microexons, we show that shared RNA secondary structures between orthologous human and chicken microexon precursor mRNAs primarily occur in regions of high sequence conservation. We find that both human and chicken neural microexons have extended functional distance between the branchpoint and the 3 splice. Structurally, branchpoint-to-splice site regions are unusually accessible and relatively unpaired compared to other exon classes. Our data suggest that microexon splicing relies on structural accessibility of the branch-point-to-splice site region, which may influence accessibility for SRRM4 binding and alleviate steric constraints for spliceosome assembly. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/699960v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@18d1f65org.highwire.dtl.DTLVardef@854fforg.highwire.dtl.DTLVardef@10463a1org.highwire.dtl.DTLVardef@128e7c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Precursor RNA structural patterns at SF3B1 mutation sensitive cryptic 3' splice sites

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3 splice site selection. SF3B1 mutation is common in myelodysplastic syndrome and other blood disorders. The most common mutation in SF3B1 is K700E, a lysine to glutamic acid change within the pre-mRNA interacting heat repeat domain. A hallmark of SF3B1 mutation is an increased use of cryptic 3 splice sites; however, the properties distinguishing SF3B1-sensitive splice junctions from other alternatively spliced junctions are unknown. We identify a subset of 192 core splice junctions that are mis-spliced with SF3B1 K700E mutation. We use our core set to test whether SF3B1-sensitive splice sites are different from control cryptic 3 splice sites via RNA structural accessibility. As a comparison, we define a set of SF3B1-resistant splice junctions with cryptic splice site use that does not change with SF3B1 K700E mutation. We find sequence differences between SF3B1-sensitive and SF3B1-resistant junctions, particularly at the cryptic sites. SF3B1-sensitive cryptic 3 splice sites are within an extended polypyrimidine tract and have lower splice site strength scores. We develop experimental RNA structure data for 83 SF3B1-sensitive junctions and 39 SF3B1-resistant junctions. We find that the pattern of structural accessibility at the NAG splicing motif in cryptic and canonical 3 splice sites is similar. In addition, this pattern can be found in both SF3B1-resistant and SF3B1-sensitive junctions. However, SF3B1-sensitive junctions have cryptic splice sites that are less structurally distinct from the canonical splice sites. In addition, SF3B1-sensitive splice junctions are overall more flexible than SF3B1-resistant junctions. Our results suggest that the SF3B1-sensitive splice junctions have unique structure and sequence properties, containing poorly differentiated, weak splice sites that lead to altered 3 splice site recognition in the presence of SF3B1 mutation.

molecular biology↗