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. 2021 Dec;18(12):2576-2593.
doi: 10.1080/15476286.2021.1932360. Epub 2021 Jun 9.

Synergistic roles for human U1 snRNA stem-loops in pre-mRNA splicing

William Martelly  1   2 , Bernice Fellows  1 , Paul Kang  3 , Ajay Vashisht  4 , James A Wohlschlegel  4 , Shalini Sharma  1
Affiliations

Synergistic roles for human U1 snRNA stem-loops in pre-mRNA splicing

William Martelly et al. RNA Biol. 2021 Dec.

Abstract

During spliceosome assembly, interactions that bring the 5' and 3' ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

Keywords: A complex; DDX39A; DDX39B; E complex; SF3A1; Spliceosome; Stem-loop; U1 snRNA; U1 snRNP; U2 snRNP; UAP56; URH49.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
Stem-loop 3 of the U1 snRNA is important for U1 function. (A) Schematic diagram of the U1 snRNP. Secondary structure and sequence of wildtype SL3 and mutations introduced into the U1 snRNA are depicted; nucleotide changes are shown in red. (B) Dup51p pre-mRNA carries a 5′-ss mutation (indicated by the red asterisk) in intron 2 that causes skipping of exon 2 in the mature transcript. (C) Primer extension analysis to monitor splicing of the minigene reporter Dup51p after cotransfections with control (pcDNA) or U1-5a plasmids expressing wildtype or mutant U1 snRNAs. The full-length and exon 2 skipped Dup51p mRNA products are depicted. The percentage of the full-length product (± s.d., n = 3) is represented in the graph below and statistical significance was determined by comparisons to the wildtype control (lane 2) (n = 3; * = p < 0.05, ** = p < 0.01). (D) RT-qPCR analysis of U1 snRNA expression in HeLa cells cotransfected with Dup51p and U1-5a variants carrying wildtype SL3 or mutations. Fold change in U1 snRNA expression was calculated relative to the pcDNA control after normalization to U2; fold change (± s.d.; n = 3) in U1 is graphed. Expression of the Dup51p reporter pre-mRNA upon cotransfection with U1-5a/SL3 mutants M1e, M1f, and M1g (lanes 7–9) appears to be reduced in this experiment. This apparent diminution, however, is not a consistent observation, as the transcript level is not reduced in Fig. 2B, lane 4
Figure 2.
Figure 2.
Combined SL3 and SL4 mutations have synergistic effects on U1 function. (A) Schematic of the SL4 secondary structures from wildtype and mutant U1 snRNAs used to create U1-SL3/SL4 double mutants. (B) Primer extension analysis to monitor splicing of the minigene reporter Dup51p after cotransfections with control or U1-5a plasmids for expression of wildtype and mutant U1 snRNA. The full-length and exon 2 skipped Dup51p mRNA products are depicted. The percentage of the full-length product (± s.d., n = 3) is graphed below and statistical significance was determined by comparisons to the wildtype control (lane 2) (n = 3; * = p < 0.05, ** = p < 0.01, *** = p < 0.001). The analysis for synergistic effects is shown in Table 1. (C) RT-qPCR analysis of U1 snRNA expression in HeLa cells cotransfected with Dup51p reporter and U1-5a variant plasmids. Fold change in U1 snRNA expression was calculated relative to the pcDNA control after normalization to U2; fold increase in U1 is graphed (± s.d., n = 3). (D) Primer extension analysis with oligonucleotide 32P-U17-26R (Supplementary Data File Table S5), showing expression of the endogenous U1 and mutant U1-5a snRNAs
Figure 3.
Figure 3.
SL3 and SL4 bind to distinct spliceosomal proteins. (A) UV crosslinking analysis for U1-SL3 interacting protein(s). HeLa nuclear extracts were incubated with 20, 40, and 80 nM 32P-U1-SL3 RNA in the presence or absence of ATP and ATP-γ-S. (B) Western analysis of proteins in wildtype and mutant U1-SL3 complexes. HeLa nuclear extracts were preincubated with 0, 0.25, 0.5, and 1.0 M NaCl prior to RNA affinity purification using biotinylated U1-SL3-WT and U1-SL3-M1g RNAs. (C) EMSAs monitoring binding of Cy5-labelled U1-SL3 (lanes 1–7) or U1-SL4 (lanes 8–14) RNAs (10 nM) in the absence and presence of GST-UAP56 (0.0625, 0.125 0.25, 0.5, 1.0, and 2.0 μM). (D) EMSAs monitoring binding of Cy5-labelled U1-SL3 (lanes 1–7) or U1-SL4 (lanes 8–14) RNAs (10 nM) to GST-SF3A1-UBL (0.03125, 0.0625, 0.125 0.25, 0.5, 1.0 μM). Displayed dose-response curves were generated by plotting the average percent of bound U1-SL3 and U1-SL4 RNA (± s.d., n = 3) versus GST-UAP56 or GST-SF3A1-UBL protein concentration and the apparent affinity constant values (KD) are reported. (E) Western analysis of proteins present in input (I) and U1 affinity purified (AP) complexes in the absence and presence of ATP-γ-S, and ATP. The intensity of the UAP56 band was normalized to that of U1-70k in the U1 and U2 complexes, and then fold change was calculated relative to the plus ATP condition (± s.d., n = 3, * = p < 0.05). (F) Western analysis of proteins present in input (I) and U2 AP complexes in the absence and presence of ATP-γ-S, and ATP. Because the signal for SF3A3 was better than SF3A1, the intensity of UAP56 band was normalized to that of SF3A3 protein in the U2 complexes, respectively, and then change was calculated relative to the plus ATP condition (± s.d., n = 3, * = p < 0.05)
Figure 4.
Figure 4.
UAP56 and SF3A1 knockdowns phenocopy SL3 and SL4 mutations, respectively. (A) Western analysis of HeLa cell lysates after treatment with non-targeting control (siNT), UAP56 targeting (siUAP56), and URH49 targeting (siURH49) siRNAs. (B) Primer extension analysis of Dup51p reporter transcripts after complementation with U1-5a variants and treatment with control siNT or siUAP56. (C) Primer extension analysis of Dup51p reporter transcripts after complementation with U1-5a variants and treatment with control siNT or siURH49. (D) Western analysis of HeLa cell lysates after treatment with control siNT or SF3A1 targeting (siSF3A1) siRNAs. (E) Primer extension analysis of Dup51p reporter transcripts after complementation with U1-5a variants and treatment with control siNT or siSF3A1. (F) Western analysis of HeLa cell lysates after treatment with control siNT or PTBP1 targeting (siPTBP1) siRNA. (G) Primer extension analysis of Dup51p reporter transcripts after complementation with U1-5a variants and treatment with control siNT or siPTBP1. Average protein expression (± s.d., n = 3) normalized to α-Tubulin is shown. Primer extension products for the full-length and exon 2 skipped Dup51p mRNA are depicted. The average percentage of the full-length product (± s.d., n = 3) is graphed below (n = 3; * = p < 0.05, ** = p < 0.01). Statistical comparisons were performed for each U1-5a snRNA tested under the siNT versus siRNA treatment conditions. Analysis for synergistic effects is shown in Table 2
Figure 5.
Figure 5.
U1-SL3 promotes the U1-SL4-SF3A1 interaction and A complex formation. (A) UV crosslinking of 32P-labelled U1-SL4 RNA in HeLa nuclear extracts in the presence of ATP. To determine the effect of free U1-SL3 and U1-SL4, the reactions were preincubated with 0.625, 1.25, 2.5, and 5.0 μM of the indicated cold stem-loop RNAs prior to addition of 32P-U1-SL4. (B) In vitro splicing of uniformly 32P-labelled AdML pre-mRNA in the absence of stem-loop RNA or in the presence of 0.625, 1.25, 2.5, and 5.0 µM wildtype U1-SL4 or U1-SL3. Splicing intermediates and products are depicted. Fold change in splicing activity is the mRNA/pre-mRNA ratio calculated relative to the no stem-loop control. Statistical analysis compared activity in the presence of U1-SL3 or U1-SL4 to the no stem-loop control (± s.d., n = 4; * = p < 0.05, ** = p < 0.01). (C) Native agarose gel analysis of ATP-dependent spliceosomal complexes assembled on uniformly 32P-labelled AdML pre-mRNA in the absence of stem-loop RNA or in the presence of 0.625, 1.25, 2.5, and 5.0 µM wildtype U1-SL4 or U1-SL3. Fold change in A complex formation is the A complex/H complex ratio calculated relative to the no stem-loop control (± s.d., n = 3, ** = p < 0.01). (D) Native agarose gel analysis of ATP-independent E complex assembled on uniformly 32P-labelled AdML pre-mRNA in the absence or in the presence of 0.625, 1.25, 2.5, and 5.0 µM wildtype U1-SL3. Fold change in E complex formation is the E complex/H complex ratio calculated relative to the no stem-loop control (± s.d., n = 3). (E) Primer extension analysis to monitor splicing of Dup51p after cotransfections with U1-5a plasmids with wildtype U1 or U1 snRNA harbouring tandem SL4 (SL4/SL4), tandem SL3 (SL3/SL3), or swapped SL3 and SL4 (SL4/SL3) structures. The full-length and exon 2 skipped Dup51p mRNA products are depicted. The percentage of the full-length product (± s.d., n = 3) is represented in the graph below and statistical significance was determined by comparisons to the wildtype control (lane 1) (n = 3; * = p < 0.05, ** = p < 0.01, *** = p < 0.001)
Figure 6.
Figure 6.
Model for the role of the U1 snRNA during early spliceosome formation. (A) During the early steps of spliceosome assembly, SL3 and SL4 of the U1 snRNA interact with the RNA helicase UAP56 and the U2 snRNP specific protein SF3A1, respectively (double headed black arrows). The SL4-SF3A1 contact bridges the 5′- and 3′-ss complexes. The SL3-UAP56 complex, directly or indirectly, promotes the SL4-SF3A1 interaction (green plus symbol) in an ATP-dependent manner, leading to enhancement of pre-mRNA splicing. (B) Disruption of the SL3-UAP56 contact by either SL3 mutations or UAP56 knockdown prevents stabilization of the SL4-SF3A1 interaction, resulting in reduced splicing. (C) Disruption of the SL4-SF3A1 interaction by either SL4 mutations or SF3A1 knockdown reduces but does not completely abrogate splicing as the SL3-UAP56 interaction can occur. In the absence of the SL4-SF3A1 contact, interaction of UAP56 with U2AF65 likely bridges the 5′- and 3′-ss complexes (grey double headed arrow) [6,55,56]. (D) The addition of excess U1-SL4 in trans competes out the interaction of SF3A1 with endogenous U1 snRNA, reducing A complex formation and inhibiting splicing in vitro [28]. (E) By contrast, addition of excess U1-SL3 in trans enhances pre-mRNA splicing by binding to endogenous UAP56. The U1-SL3-UAP56 complex promotes the SL4-SF3A1 interaction in an ATP-dependent manner, enhancing A complex formation and splicing
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