Regulation of multiple core spliceosomal proteins by alternative splicing-coupled nonsense-mediated mRNA decay - PubMed (original) (raw)

Regulation of multiple core spliceosomal proteins by alternative splicing-coupled nonsense-mediated mRNA decay

Arneet L Saltzman et al. Mol Cell Biol. 2008 Jul.

Abstract

Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2, and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions, and surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes that encode core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.

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Figures

FIG. 1.

FIG. 1.

Overlapping but distinct effects of UPF protein knockdowns on PTC-introducing AS events. (A) Western blot assays of HeLa cell lysates following siRNA-mediated UPF factor knockdowns. Western blots were probed with UPF2- or UPF3-specific antibodies (upper panels) and with calnexin- or β-actin-specific antibodies as loading controls (lower panels). Serial threefold dilutions of control lysates are shown in the left panels. An estimation of the knockdown efficiency following targeting siRNA treatment relative to the control siRNA treatment is shown below the gels (right panels). For a Western blot assay showing knockdown of UPF1, see reference . (B) The change in alternative exon inclusion level (percent skipping) is shown for AS events that introduce a PTC upon inclusion (left panel) or upon skipping (right panel). The change in percent skipping is represented by the cyan-black-yellow color scale shown and is calculated for each AS event as the percent skipping in the UPF1, UPF2, or UPF3X knockdown minus the percent skipping for the same AS event in the corresponding control siRNA treatment. Events for which we detected at least a 5% change in percent skipping upon UPF1 knockdown are shown, and rows are ordered according to the median of the percent skipping change across the three knockdowns.

FIG. 2.

FIG. 2.

Representative RT-PCR assays showing effects of UPF protein knockdown on levels of PTC-introducing alternative exons. RT-PCR assays were performed with primers specific for the constitutive exons flanking the alternative exons. The RNA samples analyzed are indicated above the lanes and correspond to cells transfected with either a control siRNA (−) or an siRNA specific for the indicated UPF factor (+). Arrows to the right of each gel indicate the expected sizes of included and skipped products. The color bar below each gel panel shows the quantification of the knockdown-dependent changes in percent skipping predicted by the microarray (upper color) and measured by RT-PCR (lower color). The change in percent skipping is shown with the same color scale as in Fig. 1B. For details on these AS events, see Table S2 in the supplemental material (PTC-upon-inclusion AS events 3, 146, 957, 557, 2315, and 2372; PTC-upon-skipping AS events 750, 1268, 76, 188, 1909, and 2914).

FIG. 3.

FIG. 3.

PTC-upon-inclusion alternative exons that show UPF1- or UPF2-dependent changes (at least a 5% difference) in inclusion level are significantly often flanked by highly conserved intronic sequences (*1, P = 2·10−4; *2, P = 3·10−2, Fisher's exact test; compare the proportions marked by asterisks to the proportion for the total group [All]). The stacked bar graph shows the proportion of PTC-upon-inclusion AS events that overlap (black) or do not overlap (white) phylogenetically conserved sequences, as identified by the phastCons algorithm (49). Overlap requires that at least 35 of the first 50 nucleotides of both the upstream and downstream intron sequences flanking the alternative exon overlap phastCons elements. “All” represents all detectable PTC-upon-inclusion events (n = 164), and “KD” represents PTC-upon-inclusion events with at least a 5% difference in the indicated direction (more inclusion or more skipping) and knockdown (UPF1KD more inclusion, n = 46; UPF2KD more inclusion, n = 16).

FIG. 4.

FIG. 4.

Conserved AS events in genes for spliceosomal proteins are more often PTC introducing than are those in genes from the control set (compare proportions marked by asterisks; P = 3·10−3, chi-square test). Conserved AS events either have flanking introns overlapping (≥35 nucleotides) phastCons conserved elements or are conserved in mouse based on an analysis of cDNAs/ESTs. PTC-introducing AS events in the spliceosomal protein set are also more often conserved than nonconserved (compare proportions marked by closed circles). Genes with more than one AS event of the same type (PTC introducing or non-PTC introducing) were only counted once.

FIG. 5.

FIG. 5.

SNRPB (also known as SmB/B′) or SMNDC1 (also known as SPF30) overexpression leads to increased levels of the respective PTC-containing (PTC+) alternative transcript. Either Flag-tagged SNRPB or SMNDC1 was transiently overexpressed in HeLa cells, and the endogenous PTC-containing transcript of SNRPB (A, left top panel) or SMNDC1 (A, right top panel) was amplified by RT-PCR with a forward primer specific for the 5′ untranslated region or an upstream exon and a reverse primer specific for the PTC-introducing alternative exon. RT-PCR amplification of conserved, PTC-containing transcripts of SF1, TRA2A, and SR140 were not affected to the same degree by the overexpression of SNRPB (A, left lower panels) or SMNDC1 (A, right lower panels). The overexpression of another Flag-tagged spliceosome-associated protein (DDX42) in parallel had little effect on the level of the SNRPB and SMNDC1 transcripts. (B) Western blot assay showing the expression of each Flag-tagged protein (left, approximate molecular sizes of markers in kilodaltons; right, arrowheads indicate expected protein sizes). Data are representative of three independent transfections, and RT-PCR assays were performed in triplicate.

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References

    1. Amrani, N., M. S. Sachs, and A. Jacobson. 2006. Early nonsense: mRNA decay solves a translational problem. Nat. Rev. Mol. Cell Biol. 7415-425. - PubMed
    1. Barbosa-Morais, N. L., M. Carmo-Fonseca, and S. Aparicio. 2006. Systematic genome-wide annotation of spliceosomal proteins reveals differential gene family expansion. Genome Res. 1666-77. - PMC - PubMed
    1. Bhattacharya, A., K. Czaplinski, P. Trifillis, F. He, A. Jacobson, and S. W. Peltz. 2000. Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay. RNA 61226-1235. - PMC - PubMed
    1. Black, D. L. 2003. Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72291-336. - PubMed
    1. Blencowe, B. J. 2006. Alternative splicing: new insights from global analyses. Cell 12637-47. - PubMed

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