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.

Czech Academy of Sciences Institute of Molecular Genetics 142 20 Prague Czech Republic

Max Planck Institute of Molecular Cell Biology and Genetics and Max Planck Institute for the Physics of Complex Systems Dresden Germany

Slovak Academy of Sciences Centre for Biosciences 840 05 Bratislava Slovak Republic

University of Southampton Faculty of Medicine Southampton SO16 6YD UK

Zobrazit více v PubMed

Wahl M.C., Will C.L., Lührmann R.. The spliceosome: design principles of a dynamic RNP machine. Cell. 2009; 136:701–718. PubMed

Ruskin B., Zamore P.D., Green M.R.. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988; 52:207–219. PubMed

Zamore P.D., Green M.R.. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc. Natl. Acad. Sci. U.S.A. 1989; 86:9243–9247. PubMed PMC

Shao C., Yang B., Wu T., Huang J., Tang P., Zhou Y., Zhou J., Qiu J., Jiang L., Li H. et al. Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome. Nat. Struct. Mol. Biol. 2014; 21:997–1005. PubMed PMC

Yoshida H., Park S.Y., Oda T., Akiyoshi T., Sato M., Shirouzu M., Tsuda K., Kuwasako K., Unzai S., Muto Y. et al. A novel 3′ splice site recognition by the two zinc fingers in the U2AF small subunit. Genes Dev. 2015; 29:1649–1660. PubMed PMC

Zarnack K., König J., Tajnik M., Martincorena I., Eustermann S., Stevant I., Reyes A., Anders S., Luscombe N.M., Ule J.. Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of PubMed PMC

Dember L.M., Kim N.D., Liu K.Q., Anderson P.. Individual RNA recognition motifs of TIA-1 and TIAR have different RNA binding specificities. J. Biol. Chem. 1996; 271:2783–2788. PubMed

Muller-McNicoll M., Botti V., de Jesus Domingues A.M., Brandl H., Schwich O.D., Steiner M.C., Curk T., Poser I., Zarnack K., Neugebauer K.M.. SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export. Genes Dev. 2016; 30:553–566. PubMed PMC

Llorian M., Schwartz S., Clark T.A., Hollander D., Tan L.Y., Spellman R., Gordon A., Schweitzer A.C., de la Grange P., Ast G. et al. Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB. Nat. Struct. Mol. Biol. 2010; 17:1114–1123. PubMed PMC

Page-McCaw P.S., Amonlirdviman K., Sharp P.A.. PUF60: a novel U2AF65-related splicing activity. RNA. 1999; 5:1548–1560. PubMed PMC

Corsini L., Hothorn M., Stier G., Rybin V., Scheffzek K., Gibson T.J., Sattler M.. Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor PUF60. J. Biol. Chem. 2009; 284:630–639. PubMed

Loerch S., Maucuer A., Manceau V., Green M.R., Kielkopf C.L.. Cancer-relevant splicing factor CAPERalpha engages the essential splicing factor SF3b155 in a specific ternary complex. J. Biol. Chem. 2014; 289:17325–17337. PubMed PMC

Crichlow G.V., Zhou H., Hsiao H.H., Frederick K.B., Debrosse M., Yang Y., Folta-Stogniew E.J., Chung H.J., Fan C., De la Cruz E.M. et al. Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition. EMBO J. 2008; 27:277–289. PubMed PMC

Loerch S., Kielkopf C.L.. Unmasking the U2AF homology motif family: a bona fide protein-protein interaction motif in disguise. RNA. 2016; 22:1795–1807. PubMed PMC

Hastings M.L., Allemand E., Duelli D.M., Myers M.P., Krainer A.R.. Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF. PLoS ONE. 2007; 2:e538. PubMed PMC

Královičová J., Knut M., Cross N.C., Vořechovský I.. Identification of U2AF(35)-dependent exons by RNA-Seq reveals a link between 3′ splice-site organization and activity of U2AF-related proteins. Nucleic Acids Res. 2015; 43:3747–3763. PubMed PMC

Liu J., He L., Collins I., Ge H., Libutti D., Li J., Egly J.M., Levens D.. The FBP interacting repressor targets TFIIH to inhibit activated transcription. Mol. Cell. 2000; 5:331–341. PubMed

Matsushita K., Tomonaga T., Shimada H., Shioya A., Higashi M., Matsubara H., Harigaya K., Nomura F., Libutti D., Levens D. et al. An essential role of alternative splicing of c-myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis. Cancer Res. 2006; 66:1409–1417. PubMed

Malz M., Bovet M., Samarin J., Rabenhorst U., Sticht C., Bissinger M., Roessler S., Bermejo J.L., Renner M., Calvisi D.F. et al. Overexpression of far upstream element (FUSE) binding protein (FBP)-interacting repressor (FIR) supports growth of hepatocellular carcinoma. Hepatology. 2014; 60:1241–1250. PubMed

Imai H., Chan E.K., Kiyosawa K., Fu X.D., Tan E.M.. Novel nuclear autoantigen with splicing factor motifs identified with antibody from hepatocellular carcinoma. J. Clin. Invest. 1993; 92:2419–2426. PubMed PMC

Dowhan D.H., Hong E.P., Auboeuf D., Dennis A.P., Wilson M.M., Berget S.M., O’Malley B.W.. Steroid hormone receptor coactivation and alternative RNA splicing by U2AF65-related proteins CAPERalpha and CAPERbeta. Mol. Cell. 2005; 17:429–439. PubMed

Ellis J.D., Lleres D., Denegri M., Lamond A.I., Cáceres J.F.. Spatial mapping of splicing factor complexes involved in exon and intron definition. J. Cell Biol. 2008; 181:921–934. PubMed PMC

Prigge J.R., Iverson S.V., Siders A.M., Schmidt E.E.. Interactome for auxiliary splicing factor U2AF(65) suggests diverse roles. Biochim. Biophys. Acta. 2009; 1789:487–492. PubMed PMC

Cazalla D., Newton K., Cáceres J.F.. A novel SR-related protein is required for the second step of Pre-mRNA splicing. Mol. Cell. Biol. 2005; 25:2969–2980. PubMed PMC

Huang S.C., Zhang H.S., Yu B., McMahon E., Nguyen D.T., Yu F.H., Ou A.C., Ou J.P., Benz E.J. Jr. Protein 4.1R Exon 16 3′ splice site activation requires coordination among TIA1, Pcbp1, and RBM39 during terminal erythropoiesis. Mol. Cell. Biol. 2017; 37:e00446-16. PubMed PMC

Shao W., Kim H.-S., Cao Y., Xu Y.-Z., Query C.C.. A U1-U2 snRNP interaction network during intron definition. Mol. Cell. Biol. 2012; 32:470–478. PubMed PMC

Stepanyuk G.A., Serrano P., Peralta E., Farr C.L., Axelrod H.L., Geralt M., Das D., Chiu H.J., Jaroszewski L., Deacon A.M. et al. UHM-ULM interactions in the RBM39-U2AF65 splicing-factor complex. Acta Crystallogr. D. Struct. Biol. 2016; 72:497–511. PubMed PMC

Kielkopf C.L., Rodionova N.A., Green M.R., Burley S.K.. A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer. Cell. 2001; 106:595–605. PubMed

Corsini L., Bonnal S., Basquin J., Hothorn M., Scheffzek K., Valcárcel J., Sattler M.. U2AF-homology motif interactions are required for alternative splicing regulation by SPF45. Nat. Struct. Mol. Biol. 2007; 14:620–629. PubMed

Mai S., Qu X., Li P., Ma Q., Cao C., Liu X.. Global regulation of alternative RNA splicing by the SR-rich protein RBM39. Biochim. Biophys. Acta. 2016; 1859:1014–1024. PubMed

Mercier I., Gonzales D.M., Quann K., Pestell T.G., Molchansky A., Sotgia F., Hulit J., Gandara R., Wang C., Pestell R.G. et al. CAPER, a novel regulator of human breast cancer progression. Cell Cycle. 2014; 13:1256–1264. PubMed PMC

Dauber A., Golzio C., Guenot C., Jodelka F.M., Kibaek M., Kjaergaard S., Leheup B., Martinet D., Nowaczyk M.J., Rosenfeld J.A. et al. SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant. Am. J. Hum. Genet. 2013; 93:798–811. PubMed PMC

El Chehadeh S., Kerstjens-Frederikse W.S., Thevenon J., Kuentz P., Bruel A.L., Thauvin-Robinet C., Bensignor C., Dollfus H., Laugel V., Riviere J.B. et al. Dominant variants in the splicing factor PUF60 cause a recognizable syndrome with intellectual disability, heart defects and short stature. Eur. J. Hum. Genet. 2016; 25:43–51. PubMed PMC

Low K.J., Ansari M., Abou Jamra R., Clarke A., El Chehadeh S., FitzPatrick D.R., Greenslade M., Henderson A., Hurst J., Keller K. et al. PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features. Eur. J. Hum. Genet. 2017; 25:552–559. PubMed PMC

Santos-Simarro F., Vallespin E., Del Pozo A., Ibanez K., Silla J.C., Fernandez L., Nevado J., Gonzalez-Pecellin H., Montano V.E.F., Martin R. et al. Eye coloboma and complex cardiac malformations belong to the clinical spectrum of PUF60 variants. Clin. Genet. 2017; 92:350–351. PubMed

Deciphering Developmental Disorders, S. Prevalence and architecture of de novo mutations in developmental disorders. Nature. 2017; 542:433–438. PubMed PMC

Královičová J., Vořechovský I.. Allele-dependent recognition of the 3′ splice site of PubMed PMC

Huranová M., Ivani I., Benda A., Poser I., Brody Y., Hof M., Shav-Tal Y., Neugebauer K.M., Staněk D.. The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. J. Cell Biol. 2010; 191:75–86. PubMed PMC

Kim D., Pertea G., Trapnell C., Pimentel H., Kelley R., Salzberg S.L.. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013; 14:R36. PubMed PMC

Langmead B., Salzberg S.L.. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012; 9:357–359. PubMed PMC

Karolchik D., Barber G.P., Casper J., Clawson H., Cline M.S., Diekhans M., Dreszer T.R., Fujita P.A., Guruvadoo L., Haeussler M. et al. The UCSC Genome Browser database: 2014 update. Nucleic Acids Res. 2014; 42:D764–D770. PubMed PMC

Anders S., Reyes A., Huber W.. Detecting differential usage of exons from RNA-seq data. Genome Res. 2012; 22:2008–2017. PubMed PMC

Lianoglou S., Garg V., Yang J.L., Leslie C.S., Mayr C.. Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression. Genes Dev. 2013; 27:2380–2396. PubMed PMC

Huang da W., Sherman B.T., Lempicki R.A.. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009; 37:1–13. PubMed PMC

Huang da W., Sherman B.T., Lempicki R.A.. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 2009; 4:44–57. PubMed

Královičová J., Gaunt T.R., Rodriguez S., Wood P.J., Day I.N.M., Vořechovský I.. Variants in the human insulin gene that affect pre-mRNA splicing: is -23 PubMed

Bailey T.L., Boden M., Buske F.A., Frith M., Grant C.E., Clementi L., Ren J., Li W.W., Noble W.S.. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009; 37:W202–W208. PubMed PMC

Ray D., Kazan H., Cook K.B., Weirauch M.T., Najafabadi H.S., Li X., Gueroussov S., Albu M., Zheng H., Yang A. et al. A compendium of RNA-binding motifs for decoding gene regulation. Nature. 2013; 499:172–177. PubMed PMC

Corvelo A., Hallegger M., Smith C.W., Eyras E.. Genome-wide association between branch point properties and alternative splicing. PLoS Comput. Biol. 2010; 6:e1001016. PubMed PMC

Yeo G., Burge C.B.. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol. 2004; 11:377–394. PubMed

Vořechovský I. Aberrant 3′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res. 2006; 34:4630–4641. PubMed PMC

Muckstein U., Tafer H., Hackermuller J., Bernhart S.H., Stadler P.F., Hofacker I.L.. Thermodynamics of RNA-RNA binding. Bioinformatics. 2006; 22:1177–1182. PubMed

Hiller M., Zhang Z., Backofen R., Stamm S.. Pre-mRNA secondary structures influence exon recognition. PLoS Genet. 2007; 3:e204. PubMed PMC

Lorenz R., Bernhart S.H., Honer Zu Siederdissen C., Tafer H., Flamm C., Stadler P.F., Hofacker I.L.. ViennaRNA Package 2.0. Algorithms Mol. Biol. 2011; 6:26. PubMed PMC

Casper J., Zweig A.S., Villarreal C., Tyner C., Speir M.L., Rosenbloom K.R., Raney B.J., Lee C.M., Lee B.T., Karolchik D. et al. The UCSC Genome Browser database: 2018 update. Nucleic Acids Res. 2017; 46:D762–D769. PubMed PMC

Rodriguez J.M., Rodriguez-Rivas J., Di Domenico T., Vazquez J., Valencia A., Tress M.L.. APPRIS 2017: principal isoforms for multiple gene sets. Nucleic Acids Res. 2017; 46:D213–D217. PubMed PMC

Wan Y., Qu K., Zhang Q.C., Flynn R.A., Manor O., Ouyang Z., Zhang J., Spitale R.C., Snyder M.P., Segal E. et al. Landscape and variation of RNA secondary structure across the human transcriptome. Nature. 2014; 505:706–709. PubMed PMC

Hiller M., Pudimat R., Busch A., Backofen R.. Using RNA secondary structures to guide sequence motif finding towards single-stranded regions. Nucleic Acids Res. 2006; 34:e117. PubMed PMC

Královičová J., Vořechovský I.. Alternative splicing of U2AF1 reveals a shared repression mechanism for duplicated exons. Nucleic Acids Res. 2017; 45:417–434. PubMed PMC

Chapman K.B., Boeke J.D.. Isolation and characterization of the gene encoding yeast debranching enzyme. Cell. 1991; 65:483–492. PubMed

Sarkar G., Sommer S.S.. The ‘megaprimer’ method of site-directed mutagenesis. BioTechniques. 1990; 8:404–407. PubMed

Královičová J., Knut M., Cross N.C., Vořechovský I.. Exon-centric regulation of ATM expression is population-dependent and amenable to antisense modification by pseudoexon targeting. Sci. Rep. 2016; 6:18741. PubMed PMC

Staněk D., Neugebauer K.M.. Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer. J. Cell Biol. 2004; 166:1015–1025. PubMed PMC

Szafranski K., Schindler S., Taudien S., Hiller M., Huse K., Jahn N., Schreiber S., Backofen R., Platzer M.. Violating the splicing rules: TG dinucleotides function as alternative 3′ splice sites in U2-dependent introns. Genome Biol. 2007; 8:R154. PubMed PMC

Gooding C., Clark F., Wollerton M., Grellscheid S.-N., Groom H., Smith C.W.. A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones. Genome Biol. 2006; 7:R1. PubMed PMC

Mercer T.R., Clark M.B., Andersen S.B., Brunck M.E., Haerty W., Crawford J., Taft R.J., Nielsen L.K., Dinger M.E., Mattick J.S.. Genome-wide discovery of human splicing branchpoints. Genome Res. 2015; 25:290–303. PubMed PMC

Královičová J., Hwang G., Asplund A.C., Churbanov A., Smith C.I., Vořechovský I.. Compensatory signals associated with the activation of human GC 5′ splice sites. Nucleic Acids Res. 2011; 39:7077–7091. PubMed PMC

Smith C.W., Nadal-Ginard B.. Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing. Cell. 1989; 56:749–758. PubMed

Denton R.M., Pullen T.J., Armstrong C.T., Heesom K.J., Rutter G.A.. Calcium-insensitive splice variants of mammalian E1 subunit of 2-oxoglutarate dehydrogenase complex with tissue-specific patterns of expression. Biochem. J. 2016; 473:1165–1178. PubMed PMC

Wang I., Hennig J., Jagtap P.K., Sonntag M., Valcárcel J., Sattler M.. Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1. Nucleic Acids Res. 2014; 42:5949–5966. PubMed PMC

Izquierdo J.M., Valcárcel J.. Two isoforms of the T-cell intracellular antigen 1 (TIA-1) splicing factor display distinct splicing regulation activities. Control of TIA-1 isoform ratio by TIA-1-related protein. J. Biol. Chem. 2007; 282:19410–19417. PubMed

Izquierdo J.M. Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R. Nucleic Acids Res. 2010; 38:8001–8014. PubMed PMC

Mayya V., Lundgren D.H., Hwang S.I., Rezaul K., Wu L., Eng J.K., Rodionov V., Han D.K.. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Sci. Signal. 2009; 2:ra46. PubMed

Bortolin M.L., Kiss T.. Human U19 intron-encoded snoRNA is processed from a long primary transcript that possesses little potential for protein coding. RNA. 1998; 4:445–454. PubMed PMC

Mikula M., Bomsztyk K., Goryca K., Chojnowski K., Ostrowski J.. Heterogeneous nuclear ribonucleoprotein (HnRNP) K genome-wide binding survey reveals its role in regulating 3′-end RNA processing and transcription termination at the early growth response 1 (EGR1) gene through XRN2 exonuclease. J. Biol. Chem. 2013; 288:24788–24798. PubMed PMC

Kimura Y., Nagata K., Suzuki N., Yokoyama R., Yamanaka Y., Kitamura H., Hirano H., Ohara O.. Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis. Proteomics. 2010; 10:3884–3895. PubMed

Wagner B.J., DeMaria C.T., Sun Y., Wilson G.M., Brewer G.. Structure and genomic organization of the human AUF1 gene: alternative pre-mRNA splicing generates four protein isoforms. Genomics. 1998; 48:195–202. PubMed

Sarkar B., Lu J.Y., Schneider R.J.. Nuclear import and export functions in the different isoforms of the AUF1/heterogeneous nuclear ribonucleoprotein protein family. J. Biol. Chem. 2003; 278:20700–20707. PubMed

Laroia G., Schneider R.J.. Alternate exon insertion controls selective ubiquitination and degradation of different AUF1 protein isoforms. Nucleic Acids Res. 2002; 30:3052–3058. PubMed PMC

Kedar V.P., Zucconi B.E., Wilson G.M., Blackshear P.J.. Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins. J. Biol. Chem. 2012; 287:5459–5471. PubMed PMC

Laguinge L., Bajenova O., Bowden E., Sayyah J., Thomas P., Juhl H.. Surface expression and CEA binding of hnRNP M4 protein in HT29 colon cancer cells. Anticancer Res. 2005; 25:23–31. PubMed

Datar K.V., Dreyfuss G., Swanson M.S.. The human hnRNP M proteins: identification of a methionine/arginine-rich repeat motif in ribonucleoproteins. Nucleic Acids Res. 1993; 21:439–446. PubMed PMC

Huelga S.C., Vu A.Q., Arnold J.D., Liang T.Y., Liu P.P., Yan B.Y., Donohue J.P., Shiue L., Hoon S., Brenner S. et al. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins. Cell Rep. 2012; 1:167–178. PubMed PMC

Lander E.S., Linton L.M., Birren B., Nusbaum C., Zody M.C., Baldwin J., Devon K., Dewar K., Doyle M., FitzHugh W. et al. Initial sequencing and analysis of the human genome. Nature. 2001; 409:860–921. PubMed

Gal-Mark N., Schwartz S., Ram O., Eyras E., Ast G.. The pivotal roles of TIA proteins in 5′ splice-site selection of alu exons and across evolution. PLoS Genet. 2009; 5:e1000717. PubMed PMC

Quentin Y. Emergence of master sequences in families of retroposons derived from 7sl RNA. Genetica. 1994; 93:203–215. PubMed

Levy A., Sela N., Ast G.. TranspoGene and microTranspoGene: transposed elements influence on the transcriptome of seven vertebrates and invertebrates. Nucleic Acids Res. 2008; 36:D47–D52. PubMed PMC

Cukier C.D., Hollingworth D., Martin S.R., Kelly G., Diaz-Moreno I., Ramos A.. Molecular basis of FIR-mediated c-myc transcriptional control. Nat. Struct. Mol. Biol. 2010; 17:1058–1064. PubMed PMC

Kuo P.H., Chiang C.H., Wang Y.T., Doudeva L.G., Yuan H.S.. The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids. Nucleic Acids Res. 2014; 42:4712–4722. PubMed PMC

Skříšovská L., Bourgeois C.F., Stefl R., Grellscheid S.N., Kister L., Wenter P., Elliott D.J., Stevenin J., Allain F.H.. The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction. EMBO Rep. 2007; 8:372–379. PubMed PMC

Dehouck Y., Kwasigroch J.M., Gilis D., Rooman M.. PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics. 2011; 12:151. PubMed PMC

Taggart A.J., DeSimone A.M., Shih J.S., Filloux M.E., Fairbrother W.G.. Large-scale mapping of branchpoints in human pre-mRNA transcripts in vivo. Nat. Struct. Mol. Biol. 2012; 19:719–721. PubMed PMC

Xue Y., Zhou Y., Wu T., Zhu T., Ji X., Kwon Y.S., Zhang C., Yeo G., Black D.L., Sun H. et al. Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. Mol. Cell. 2009; 36:996–1006. PubMed PMC

Poleev A., Hartmann A., Stamm S.. A trans-acting factor, isolated by the three-hybrid system, that influences alternative splicing of the amyloid precursor protein minigene. Eur. J. Biochem. 2000; 267:4002–4010. PubMed

Lin C.L., Taggart A.J., Lim K.H., Cygan K.J., Ferraris L., Creton R., Huang Y.T., Fairbrother W.G.. RNA structure replaces the need for U2AF2 in splicing. Genome Res. 2016; 26:12–23. PubMed PMC

Lambert N.J., Robertson A.D., Burge C.B.. RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins. Methods Enzymol. 2015; 558:465–493. PubMed PMC

Kennedy C.F., Kramer A., Berget S.M.. A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point. Mol. Cell. Biol. 1998; 18:5425–5434. PubMed PMC

Zhang W.J., Wu J.Y.. Functional properties of p54, a novel SR protein active in constitutive and alternative splicing. Mol. Cell. Biol. 1996; 16:5400–5408. PubMed PMC

McCullough A.J., Berget S.M.. G triplets located throughout a class of small vertebrate introns enforce intron borders and regulate splice site selection. Mol. Cell. Biol. 1997; 17:4562–4571. PubMed PMC

Yeo G., Hoon S., Venkatesh B., Burge C.B.. Variation in sequence and organization of splicing regulatory elements in vertebrate genes. Proc. Natl. Acad. Sci. U.S.A. 2004; 101:15000–15005. PubMed PMC

Kelley D.R., Hendrickson D.G., Tenen D., Rinn J.L.. Transposable elements modulate human RNA abundance and splicing via specific RNA-protein interactions. Genome Biol. 2014; 15:537. PubMed PMC

Lev-Maor G., Sorek R., Shomron N., Ast G.. The birth of an alternatively spliced exon: 3′ splice-site selection in PubMed

Cisse I.I., Kim H., Ha T.. A rule of seven in Watson-Crick base-pairing of mismatched sequences. Nat. Struct. Mol. Biol. 2012; 19:623–627. PubMed PMC

Buratti E., Dhir A., Lewandowska M.A., Baralle F.E.. RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events. Nucleic Acids Res. 2007; 35:4369–4383. PubMed PMC

Královičová J., Patel A., Searle M., Vořechovský I.. The role of short RNA loops in recognition of a single-hairpin exon derived from a mammalian-wide interspersed repeat. RNA Biol. 2015; 12:54–69. PubMed PMC

Nozu K., Iijima K., Igarashi T., Yamada S., Královičová J., Nozu Y., Yamamura T., Minamikawa S., Morioka I., Ninchoji T. et al. A birth of bipartite exon by intragenic deletion. Mol. Genet. Genomic Med. 2017; 5:287–294. PubMed PMC

Maniatis T., Reed R.. An extensive network of coupling among gene expression machines. Nature. 2002; 416:499–506. PubMed

Kaida D., Berg M.G., Younis I., Kasim M., Singh L.N., Wan L., Dreyfuss G.. U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation. Nature. 2010; 468:664–668. PubMed PMC

Wang Z., Kayikci M., Briese M., Zarnack K., Luscombe N.M., Rot G., Zupan B., Curk T., Ule J.. iCLIP predicts the dual splicing effects of TIA-RNA interactions. PLoS Biol. 2010; 8:e1000530. PubMed PMC

Singh N.N., Seo J., Ottesen E.W., Shishimorova M., Bhattacharya D., Singh R.N.. TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy. Mol. Cell. Biol. 2011; 31:935–954. PubMed PMC

Forch P., Puig O., Martinez C., Séraphin B., Valcárcel J.. The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5′ splice sites. EMBO J. 2002; 21:6882–6892. PubMed PMC

Manceau V., Swenson M., Le Caer J.P., Sobel A., Kielkopf C.L., Maucuer A.. Major phosphorylation of SF1 on adjacent Ser-Pro motifs enhances interaction with U2AF65. FEBS J. 2006; 273:577–587. PubMed PMC

Kupfer D.M., Drabenstot S.D., Buchanan K.L., Lai H., Zhu H., Dyer D.W., Roe B.A., Murphy J.W.. Introns and splicing elements of five diverse fungi. Eukaryot. Cell. 2004; 3:1088–1100. PubMed PMC

Afroz T., Cienikova Z., Cléry A., Allain F.H.. One, two, three, four! How multiple RRMs read the genome sequence. Methods Enzymol. 2015; 558:235–278. PubMed

Kim S.W., Taggart A.J., Heintzelman C., Cygan K.J., Hull C.G., Wang J., Shrestha B., Fairbrother W.G.. Widespread intra-dependencies in the removal of introns from human transcripts. Nucleic Acids Res. 2017; 45:9503–9513. PubMed PMC

Yoshida K., Sanada M., Shiraishi Y., Nowak D., Nagata Y., Yamamoto R., Sato Y., Sato-Otsubo A., Kon A., Nagasaki M. et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011; 478:64–69. PubMed

Alsafadi S., Houy A., Battistella A., Popova T., Wassef M., Henry E., Tirode F., Constantinou A., Piperno-Neumann S., Roman-Roman S. et al. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat. Commun. 2016; 7:10615. PubMed PMC

Castello A., Fischer B., Hentze M.W., Preiss T.. RNA-binding proteins in Mendelian disease. Trends Genet. 2013; 29:318–327. PubMed

McCann K.L., Teramoto T., Zhang J., Tanaka Hall T.M., Baserga S.J.. The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome. Elife. 2016; 5:e16381. PubMed PMC

Buratti E., Chivers M.C., Hwang G., Vořechovský I.. DBASS3 and DBASS5: databases of aberrant 3′ and 5′ splice sites in human disease genes. Nucleic Acids Res. 2011; 39:D86–D91. PubMed PMC

Huie M.L., Chen A.S., Tsujino S., Shanske S., DiMauro S., Engel A.G., Hirschhorn R.. Aberrant splicing in adult onset glycogen storage disease type II (GSDII): molecular identification of an IVS1 (-13T→G) mutation in a majority of patients and a novel IVS10 (+1GT→CT) mutation. Hum. Mol. Genet. 1994; 3:2231–2236. PubMed

Loudianos G., Lovicu M., Dessi V., Tzetis M., Kanavakis E., Zancan L., Zelante L., Galvez-Galvez C., Cao A.. Abnormal mRNA splicing resulting from consensus sequence splicing mutations of PubMed

Wu J., Li C., Zhao S., Mao B.. Differential expression of the Brunol/CELF family genes during Xenopus laevis early development. Int. J. Dev. Biol. 2010; 54:209–214. PubMed

Yang Y., Mahaffey C.L., Berube N., Maddatu T.P., Cox G.A., Frankel W.N.. Complex seizure disorder caused by Brunol4 deficiency in mice. PLoS Genet. 2007; 3:e124. PubMed PMC

Porath B., Gainullin V.G., Cornec-Le Gall E., Dillinger E.K., Heyer C.M., Hopp K., Edwards M.E., Madsen C.D., Mauritz S.R., Banks C.J. et al. Mutations in GANAB, encoding the glucosidase II alpha subunit, cause autosomal-dominant polycystic kidney and liver disease. Am. J. Hum. Genet. 2016; 98:1193–1207. PubMed PMC

Besse W., Dong K., Choi J., Punia S., Fedeles S.V., Choi M., Gallagher A.R., Huang E.B., Gulati A., Knight J. et al. Isolated polycystic liver disease genes define effectors of polycystin-1 function. J. Clin. Invest. 2017; 127:1772–1785. PubMed PMC

Pelletier M.F., Marcil A., Sevigny G., Jakob C.A., Tessier D.C., Chevet E., Menard R., Bergeron J.J., Thomas D.Y.. The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo. Glycobiology. 2000; 10:815–827. PubMed

Satoh T., Toshimori T., Yan G., Yamaguchi T., Kato K.. Structural basis for two-step glucose trimming by glucosidase II involved in ER glycoprotein quality control. Sci. Rep. 2016; 6:20575. PubMed PMC

Ke S., Shang S., Kalachikov S.M., Morozova I., Yu L., Russo J.J., Ju J., Chasin L.A.. Quantitative evaluation of all hexamers as exonic splicing elements. Genome Res. 2011; 21:1360–1374. PubMed PMC

Goren A., Ram O., Amit M., Keren H., Lev-Maor G., Vig I., Pupko T., Ast G.. Comparative analysis identifies exonic splicing regulatory sequences - The complex definition of enhancers and silencers. Mol. Cell. 2006; 22:769–781. PubMed

Restriction of an intron size en route to endothermy

Cancer-Associated Substitutions in RNA Recognition Motifs of PUF60 and U2AF65 Reveal Residues Required for Correct Folding and 3' Splice-Site Selection