Multiple protein–protein interactions converging on the Prp38 protein during activation of the human spliceosome

Schütze, Tonio; Ulrich, Alexander; Apelt, Luise; Will, Cindy L.; Bartlick, Natascha; Seeger, M.A.; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C.

doi:10.1261/rna.054296.115

Cited by 26 publications

(41 citation statements)

References 44 publications

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“…Sc Spp381 shares the ability of hs MFAP1 to bind Prp38, as shown by Y2H analyses [21, 43]. We confirmed this interaction with isolated, recombinant, wild type sc Prp38 and sc Spp381 proteins that co-migrated on a gel filtration column (Fig.…”

Section: Resultssupporting

confidence: 77%

“…In human, the group of B-specific proteins includes Prp38, Snu23, microfibrillar-associated protein 1 (MFAP1), suppressor of mec-8 and unc-52 protein homolog 1 (Smu1), Arg-Glu/Asp-repeat-containing protein (RED), formin-binding protein 21 (FBP21), 38 kDa nuclear protein containing a WW domain (NPW38), NPW38-binding protein (NPW38BP) and ubiquitin-like protein 5 (UBL5) [20]. Homo sapiens ( hs ) Prp38 has acquired a veritable, C-terminal arginine-serine-rich (RS) domain, a hallmark of the splicing regulatory serine-arginine-rich (SR) proteins that are largely lacking in yeast [21]. UBL5, also called Hub1, was the first splicing factor that was found to be involved in alterative splicing in human [22] as well as in a rare case of alternative splicing in yeast [23].…”

Section: Introductionmentioning

confidence: 99%

“…Later, the protein was found in spliceosome preparations [31], was shown to interact with Prp38 in pull-down experiments and to be required for pre-mRNA processing [32]. Interactions between MFAP1 and other B-specific proteins were identified by yeast two-hybrid (Y2H) [21, 33] and in vitro binding studies [21, 34]. Due to its elongated, solvent exposed nature and predicted dense array of short protein binding motifs, MFAP1 was suggested to act as a scaffold or ruler that engages multiple binding partners [34].…”

Section: Introductionmentioning

confidence: 99%

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Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor

Ulrich

Wahl

2017

BMC Evol Biol

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BackgroundPre-mRNA splicing involves the stepwise assembly of a pre-catalytic spliceosome, followed by its catalytic activation, splicing catalysis and disassembly. Formation of the pre-catalytic spliceosomal B complex involves the incorporation of the U4/U6.U5 tri-snRNP and of a group of non-snRNP B-specific proteins. While in Saccharomyces cerevisiae the Prp38 and Snu23 proteins are recruited as components of the tri-snRNP, metazoan orthologs of Prp38 and Snu23 associate independently of the tri-snRNP as members of the B-specific proteins. The human spliceosome contains about 80 proteins that lack obvious orthologs in yeast, including most of the B-specific proteins apart from Prp38 and Snu23. Conversely, the tri-snRNP protein Spp381 is one of only five S. cerevisiae splicing factors without a known human ortholog.ResultsUsing InParanoid, a state-of-the-art method for ortholog inference between pairs of species, and systematic BLAST searches we identified the human B-specific protein MFAP1 as a putative ortholog of the S. cerevisiae tri-snRNP protein Spp381. Bioinformatics revealed that MFAP1 and Spp381 share characteristic structural features, including intrinsic disorder, an elongated shape, solvent exposure of most residues and a trend to adopt α-helical structures. In vitro binding studies showed that human MFAP1 and yeast Spp381 bind their respective Prp38 proteins via equivalent interfaces and that they cross-interact with the Prp38 proteins of the respective other species. Furthermore, MFAP1 and Spp381 both form higher-order complexes that additionally include Snu23, suggesting that they are parts of equivalent spliceosomal sub-complexes. Finally, similar to yeast Spp381, human MFAP1 partially rescued a growth defect of the temperature-sensitive mutant yeast strain prp38-1.ConclusionsHuman B-specific protein MFAP1 structurally and functionally resembles the yeast tri-snRNP-specific protein Spp381 and thus qualifies as its so far missing ortholog. Our study indicates that the yeast Snu23-Prp38-Spp381 triple complex was evolutionarily reprogrammed from a tri-snRNP-specific module in yeast to the B-specific Snu23-Prp38-MFAP1 module in metazoa, affording higher flexibility in spliceosome assembly and thus, presumably, in splicing regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0923-1) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 77%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor

Ulrich

Wahl

2017

BMC Evol Biol

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“…Prp38, the yeast homolog of PRPF38A, is required for catalytic activation of the tri-snRNP complex (24,25). Thus, PRPF8 and PRPF38A are both important for tri-snRNP function.…”

Section: Resultsmentioning

confidence: 99%

“…PRPF38A interacts with 28 other RNA splicing proteins, including tri-snRNP proteins, A complex proteins, B complex proteins, proteins involved in B complex activation, and U2 snRNA-interacting proteins. As such, PRPF38A is one of the largest protein-protein-interaction hubs of the human spliceosome (25). …”

Section: Discussionmentioning

confidence: 99%

Basal-A Triple-Negative Breast Cancer Cells Selectively Rely on RNA Splicing for Survival

Chan

Sridhar

Kirchner

et al. 2017

Molecular Cancer Therapeutics

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Prognosis of triple-negative breast cancer (TNBC) remains poor. To identify shared and selective vulnerabilities of basal-like TNBC, the most common TNBC subtype, a directed siRNA lethality screen was performed in 7 human breast cancer cell lines, focusing on 154 previously identified dependency genes of one TNBC line. Thirty common dependency genes were identified, including multiple proteasome and RNA splicing genes, especially those associated with the U4/U6.U5 tri-snRNP complex (e.g., PRPF8, PRPF38A). PRPF8 or PRPF38A knockdown or the splicing modulator E7107 led to widespread intronic retention and altered splicing of transcripts involved in multiple basal-like TNBC dependencies, including protein homeostasis, mitosis and apoptosis. E7107 treatment suppressed the growth of basal-A TNBC cell line and patient-derived basal-like TNBC xenografts at a well-tolerated dose. The anti-tumor response was enhanced by adding the proteasome inhibitor bortezomib. Thus, inhibiting both splicing and the proteasome might be an effective approach for treating basal-like TNBC.

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Identifying HIPK1 as Target of miR‐22‐3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen

Inwood

Buehler

Betenbaugh

et al. 2017

Biotechnology Journal

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Protein expression from human embryonic kidney cells (HEK 293) is an important tool for structural and clinical studies. It is previously shown that microRNAs (small, noncoding RNAs) are effective means for improved protein expression from these cells, and by conducting a high-throughput screening of the human microRNA library, several microRNAs are identified as potential candidates for improving expression. From these, miR-22-3p is chosen for further study since it increased the expression of luciferase, two membrane proteins and a secreted fusion protein with minimal effect on the cells' growth and viability. Since each microRNA can interact with several gene targets, it is of interest to identify the repressed genes for understanding and exploring the improved expression mechanism for further implementation. Here, the authors describe a novel approach for identification of the target genes by integrating the differential gene expression analysis with information obtained from our previously conducted high-throughput siRNA screening. The identified genes were validated as being involved in improving luciferase expression by using siRNA and qRT-PCR. Repressing the target gene, HIPK1, is found to increase luciferase and GPC3 expression 3.3- and 2.2-fold, respectively.

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Multiple protein–protein interactions converging on the Prp38 protein during activation of the human spliceosome

Cited by 26 publications

References 44 publications

Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor

Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor

Basal-A Triple-Negative Breast Cancer Cells Selectively Rely on RNA Splicing for Survival

Identifying HIPK1 as Target of miR‐22‐3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen

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