PUF60-activated exons uncover altered 3′ splice-site selection by germline missense mutations in a single RRM

Abstract

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.

U2 AF 65 assemblies drive sequence‐specific splice site recognition

“…Other reports have documented similar observations: Fu and colleagues have proposed that U2AF 65 could have a long distance negative effect on spliceosome assembly by an unknown mechanism . Vorechovski and colleagues suggested that U2AF 65 or CAPERα could facilitate the recruitment of inhibitory pyrimidine‐binding proteins on long AG exclusions zones . However, U2AF 65 is more known to be essential for spliceosome assembly .…”

“…In contrast with U2AF 65 and CAPERα, our analysis of RNA‐Seq data point to a negative relationship of Splicing Changes upon PUF60 knockdown with ΔSPY scores in HEK293T cells and a less pronounced one in K562 cells (Fig EV2C). Similarly, Vorechovsky and colleagues observed opposite effects of U2AF and PUF60 knockdown on cassette exons preceded by extended uridine‐rich regions in HEK293T cells . Further investigations are needed to state the significance of this overlap between PUF60‐repressed and U2AF 65 ‐activated exons and its molecular basis.…”

“…We found a significant positive correlation of "Splicing Changes" upon knockdown of CAPERa and U2AF 65 with the DSPY scores ( Fig 4F, Spearman rho = 0.40, P = 1.6E-03 and rho = 0.33, P = 8.8E-03, respectively). To generalize these observations, we performed similar analyses using available RNA-Seq data for K562 [43], HeLa [44], MCF7 [42], and HEK293T [45] cells. We aligned reads to our library of sequences corresponding to 5,629 cassette exon inclusions and exclusions.…”

Novel diagnosis and therapy for hepatoma targeting HBV-related carcinogenesis through alternative splicing of FIR (PUF60)/FIRΔexon2

“…Rare disease CHARGE syndrome [59] Phenotypic variability of genetic diseases [59] Verheji syndrome [59,60] developmental delay, intelllectual disability, microcephaly, craniofacial, renal and cardiac defects [58,59] Eye coloboma and complex cardiac malformations [59,61] atrioventricular septal defect and hypoplastic aortic arch, facial dysmorphism, microretrognathia, dysmorphic ears, clinodactyly of the 5th digit on both hands, mild rocker bottom feet and abnormal third sacral vertebra [61,62] microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C > T variant leading to a p.His169Tyr change in PUF60. (PUF60 deficiency) [63][64][65] Figure 5. BK697 inhibit far upstream element-binding protein-interacting repressor Δexon2 (FIRΔexon2) that is considered as a dominant negative of FIR.…”

Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing

“…Interestingly, SR protein binding to the WT substrates appeared to maintain, enhance, or weakly suppress the flexibility of the PPT region while SR protein binding to the EH3+4 mutant appeared to strongly suppress the flexibility of this region ( Figure 6B): the p-value of the difference in ΔSHAPE reactivity in the PPT and its flanking areas [150-250 nt] of β-globin WT+SRSF1 and β-globin EH3+4+SRSF1 mutant is 3.90E-08; and that of β-globin WT+SRSF2 and β-globin EH3+4+SRSF2 is 1.88E-09. This might be reflective of the requirement of a more single-stranded PPT region for splicing (57).…”