Smu1 and RED are required for activation of spliceosomal B complexes assembled on short introns

Abstract

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA’s 5′ splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5′ and 3′ regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5′SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with the splicing of short introns.

Influenza virus infection induces widespread alterations of host cell splicing

“…We identified 11 571 ASEs corresponding to 4570 genes, 14% of which are non-annotated splicing events, that undergo marked (|ΔPSI| ≥ 10%) and significant ( P ≤ 0.05) differential regulation upon depletion of RED ( Supplementary Figure S10B and Supplementary Table S7 ). A clear trend for decreased exon inclusion and increased IR was observed in RED-depleted cells ( Supplementary Figure S10C ), in agreement with previously published studies ( 31 , 32 ).…”

“…Poly(A)-tailed RNAs were extracted and subjected to HiSeq2500 Illumina sequencing, and splicing changes induced by RED depletion were analyzed using the same pipeline as described in Figure 1A . PCA indicated that the control siRNA had no effect on splicing and that RED depletion accounted for 45% of the total variance observed in splicing and up to 63% of the total variance observed in the subset of IR events ( Supplementary Figure S10A ), in agreement with the specific requirement of RED–SMU1 for the splicing of short introns ( 31 ). IAV infection accounted for a lower percentage of the variance (16%) along a clearly separate axis.…”

SPF45/RBM17-dependent, but not U2AF-dependent, splicing in a distinct subset of human short introns

“…Here we have just described one distinct subset of human SPF45-dependent short introns. Most recently, Smu1 and RED proteins were shown to activate spliceosomal B complexes assembled on human short introns (Keiper et al, 2019). The distance between the 5′ splice site and branch site need to be sufficiently short for Smu1/RED-dependent splicing, whereas in contrast, we clearly showed that this distance per se is not responsible for SPF45-dependent splicing (Figure 2).…”

SPF45/RBM17-dependent, but not U2AF-dependent, splicing in human short introns

“…Here, we have just described one distinct subset of human SPF45-dependent short introns. Most recently, Smu1 and RED proteins were shown to activate spliceosomal B complexes assembled on human short introns 38 . The distance between the 5′ splice site and branch site need to be sufficiently short for Smu1/RED-dependent splicing, whereas in contrast, we clearly showed that this distance per se is not responsible for SPF45-dependent splicing (Fig.…”