Ca2+-insensitive and -sensitive E1 subunits of the 2-oxoglutarate dehydrogenase complex (OGDHC) regulate tissue-specific NADH and ATP supply by mutually exclusive OGDH exons 4a and 4b. Here we show that their splicing is enforced by distant lariat branch points (dBPs) located near the 5' splice site of the intervening intron. dBPs restrict the intron length and prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed by a single dominant dBP in anamniotes that expanded into a conserved constellation of four dBP adenines in amniotes. The amniote clusters exhibit taxon-specific usage of individual dBPs, reflecting accessibility of their extended motifs within a stable RNA hairpin rather than U2 snRNA:dBP base-pairing. The dBP expansion took place in early terrestrial species and was followed by a uridine enrichment of large downstream polypyrimidine tracts in mammals. The dBP-protected megatracts permit reciprocal regulation of exon 4a and 4b by uridine-binding proteins, including TIA-1/TIAR and PUF60, which promote U1 and U2 snRNP recruitment to the 5' splice site and BP, respectively, but do not significantly alter the relative dBP usage. We further show that codons for residues critically contributing to protein binding sites for Ca2+ and other divalent metals confer the exon inclusion order that mirrors the Irving-Williams affinity series, linking the evolution of auxiliary splicing motifs in exons to metallome constraints. Finally, we hypothesize that the dBP-driven selection for Ca2+-dependent ATP provision by E1 facilitated evolution of endothermy by optimizing the aerobic scope in target tissues.
- MeSH
- alternativní sestřih * MeSH
- exony MeSH
- HEK293 buňky MeSH
- introny * MeSH
- ketoglutarátdehydrogenasový komplex genetika metabolismus MeSH
- lidé MeSH
- messenger RNA chemie metabolismus MeSH
- místa sestřihu RNA MeSH
- molekulární evoluce MeSH
- obratlovci genetika MeSH
- prekurzory RNA chemie metabolismus MeSH
- protein - isoformy genetika metabolismus MeSH
- rozptýlené repetitivní sekvence MeSH
- sestřihové faktory metabolismus MeSH
- spliceozomy metabolismus MeSH
- termoregulace genetika MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.
- MeSH
- aminokyselinové motivy MeSH
- exony * MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- heterogenní jaderné ribonukleoproteiny metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- krátké rozptýlené jaderné elementy MeSH
- lidé MeSH
- malý jaderný ribonukleoprotein U1 metabolismus MeSH
- missense mutace * MeSH
- místa sestřihu RNA * MeSH
- proteiny vázající RNA metabolismus MeSH
- represorové proteiny chemie nedostatek metabolismus MeSH
- sekvenční analýza RNA MeSH
- sestřihové faktory chemie nedostatek metabolismus MeSH
- sestřihový faktor U2AF MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH