Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion.
Journal
Nature biotechnology
ISSN: 1546-1696
Titre abrégé: Nat Biotechnol
Pays: United States
ID NLM: 9604648
Informations de publication
Date de publication:
02 Jan 2024
02 Jan 2024
Historique:
received:
20
05
2023
accepted:
29
09
2023
medline:
4
1
2024
pubmed:
4
1
2024
entrez:
3
1
2024
Statut:
aheadofprint
Résumé
RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.
Identifiants
pubmed: 38168984
doi: 10.1038/s41587-023-02014-0
pii: 10.1038/s41587-023-02014-0
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NHGRI NIH HHS
ID : R01 HG004659
Pays : United States
Organisme : NHGRI NIH HHS
ID : U24 HG009889
Pays : United States
Organisme : NCI NIH HHS
ID : U54 CA209891
Pays : United States
Informations de copyright
© 2024. The Author(s).
Références
Gerstberger, S., Hafner, M. & Tuschl, T. A census of human RNA-binding proteins. Nat. Rev. Genet. 15, 829–845 (2014).
doi: 10.1038/nrg3813
pubmed: 25365966
Queiroz, R. M. L. et al. Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS). Nat. Biotechnol. 37, 169–178 (2019).
doi: 10.1038/s41587-018-0001-2
pubmed: 30607034
pmcid: 6591131
Jiang, W. & Chen, L. Alternative splicing: human disease and quantitative analysis from high-throughput sequencing. Comput. Struct. Biotechnol. J. 19, 183–195 (2021).
pubmed: 33425250
doi: 10.1016/j.csbj.2020.12.009
Wheeler, E. C. et al. Integrative RNA-omics discovers GNAS alternative splicing as a phenotypic driver of splicing factor–mutant neoplasms. Cancer Discov. 12, 836–855 (2022).
pubmed: 34620690
pmcid: 8904276
doi: 10.1158/2159-8290.CD-21-0508
Bradley, R. K. & Anczuków, O. RNA splicing dysregulation and the hallmarks of cancer. Nat. Rev. Cancer 23, 135–155 (2023).
Scotti, M. M. & Swanson, M. S. RNA mis-splicing in disease. Nat. Rev. Genet. 17, 19–32 (2016).
pubmed: 26593421
doi: 10.1038/nrg.2015.3
Rogalska, M. E., Vivori, C. & Valcárcel, J. Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects. Nat. Rev. Genet. 24, 251–269 (2022).
Zheng, S., Damoiseaux, R., Chen, L. & Black, D. L. A broadly applicable high-throughput screening strategy identifies new regulators of Dlg4 (Psd-95) alternative splicing. Genome Res. 23, 998–1007 (2013).
pubmed: 23636947
pmcid: 3668367
doi: 10.1101/gr.147546.112
Moore, M. J., Wang, Q., Kennedy, C. J. & Silver, P. A. An alternative splicing network links cell-cycle control to apoptosis. Cell 142, 625–636 (2010).
pubmed: 20705336
pmcid: 2924962
doi: 10.1016/j.cell.2010.07.019
Tejedor, J. R., Papasaikas, P. & Valcárcel, J. Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis. Mol. Cell 57, 23–38 (2015).
pubmed: 25482508
doi: 10.1016/j.molcel.2014.10.029
Sun, S., Zhang, Z., Fregoso, O. & Krainer, A. R. Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2. RNA 18, 274–283 (2012).
pubmed: 22184459
pmcid: 3264914
doi: 10.1261/rna.030486.111
Yeo, G. W. et al. An RNA code for the FOX2 splicing regulator revealed by mapping RNA–protein interactions in stem cells. Nat. Struct. Mol. Biol. 16, 130–137 (2009).
pubmed: 19136955
pmcid: 2735254
doi: 10.1038/nsmb.1545
Lovci, M. T. et al. Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges. Nat. Struct. Mol. Biol. 20, 1434–1442 (2013).
pubmed: 24213538
pmcid: 3918504
doi: 10.1038/nsmb.2699
Barash, Y. et al. Deciphering the splicing code. Nature 465, 53–59 (2010).
pubmed: 20445623
doi: 10.1038/nature09000
Tycko, J. et al. High-throughput discovery and characterization of human transcriptional effectors. Cell 183, 2020–2035 (2020).
pubmed: 33326746
pmcid: 8178797
doi: 10.1016/j.cell.2020.11.024
Luo, E.-C. et al. Large-scale tethered function assays identify factors that regulate mRNA stability and translation. Nat. Struct. Mol. Biol. 27, 989–1000 (2020).
pubmed: 32807991
pmcid: 8221285
doi: 10.1038/s41594-020-0477-6
Bos, T. J., Nussbacher, J. K., Aigner, S. & Yeo, G. W. Tethered function assays as tools to elucidate the molecular roles of RNA-binding proteins. In RNA Processing (ed. Yeo, G. W.) 61–88 (Springer, 2016).
Wang, Y., Cheong, C.-G., Tanaka Hall, T. M. & Wang, Z. Engineering splicing factors with designed specificities. Nat. Methods 6, 825–830 (2009).
pubmed: 19801992
pmcid: 2963066
doi: 10.1038/nmeth.1379
Du, M., Jillette, N., Zhu, J. J., Li, S. & Cheng, A. W. CRISPR artificial splicing factors. Nat. Commun. 11, 2973 (2020).
pubmed: 32532987
pmcid: 7293279
doi: 10.1038/s41467-020-16806-4
Leclair, N. K. et al. Poison exon splicing regulates a coordinated network of SR protein expression during differentiation and tumorigenesis. Mol. Cell 80, 648–665 (2020).
pubmed: 33176162
pmcid: 7680420
doi: 10.1016/j.molcel.2020.10.019
Liu, F. & Gong, C.-X. Tau exon 10 alternative splicing and tauopathies. Mol. Neurodegener. 3, 8 (2008).
pubmed: 18616804
pmcid: 2483273
doi: 10.1186/1750-1326-3-8
Popp, M. W. & Maquat, L. E. Leveraging rules of nonsense-mediated mRNA decay for genome engineering and personalized medicine. Cell 165, 1319–1322 (2016).
pubmed: 27259145
pmcid: 4924582
doi: 10.1016/j.cell.2016.05.053
Chamieh, H., Ballut, L., Bonneau, F. & Le Hir, H. NMD factors UPF2 and UPF3 bridge UPF1 to the exon junction complex and stimulate its RNA helicase activity. Nat. Struct. Mol. Biol. 15, 85–93 (2008).
pubmed: 18066079
doi: 10.1038/nsmb1330
Boehm, V. et al. SMG5-SMG7 authorize nonsense-mediated mRNA decay by enabling SMG6 endonucleolytic activity. Nat. Commun. 12, 3965 (2021).
pubmed: 34172724
pmcid: 8233366
doi: 10.1038/s41467-021-24046-3
Binder, J. X. et al. COMPARTMENTS: unification and visualization of protein subcellular localization evidence. Database 2014, bau012 (2014).
pubmed: 24573882
pmcid: 3935310
doi: 10.1093/database/bau012
Bondy-Chorney, E. et al. Staufen1 regulates multiple alternative splicing events either positively or negatively in DM1 indicating its role as a disease modifier. PLoS Genet. 12, e1005827 (2016).
pubmed: 26824521
pmcid: 4733145
doi: 10.1371/journal.pgen.1005827
Bondy-Chorney, E., Crawford Parks, T. E., Ravel-Chapuis, A., Jasmin, B. J. & Côté, J. Staufen1s role as a splicing factor and a disease modifier in myotonic dystrophy type I. Rare Dis. 4, e1225644 (2016).
pubmed: 27695661
pmcid: 5027583
doi: 10.1080/21675511.2016.1225644
Van Nostrand, E. L. et al. A large-scale binding and functional map of human RNA-binding proteins. Nature 583, 711–719 (2020).
pubmed: 32728246
pmcid: 7410833
doi: 10.1038/s41586-020-2077-3
Ambrozková, M. et al. The fission yeast ortholog of the coregulator SKIP interacts with the small subunit of U2AF. Biochem. Biophys. Res. Commun. 284, 1148–1154 (2001).
pubmed: 11414703
doi: 10.1006/bbrc.2001.5108
Selenko, P. et al. Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP. Mol. Cell 11, 965–976 (2003).
pubmed: 12718882
doi: 10.1016/S1097-2765(03)00115-1
Matera, A. G. & Wang, Z. A day in the life of the spliceosome. Nat. Rev. Mol. Cell Biol. 15, 108–121 (2014).
pubmed: 24452469
pmcid: 4060434
doi: 10.1038/nrm3742
Cvitkovic, I. & Jurica, M. S. Spliceosome Database: a tool for tracking components of the spliceosome. Nucleic Acids Res. 41, D132–D141 (2013).
pubmed: 23118483
doi: 10.1093/nar/gks999
Chen, Y.-I. G. et al. Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors. Nucleic Acids Res. 35, 3928–3944 (2007).
pubmed: 17537823
pmcid: 1919476
doi: 10.1093/nar/gkm347
Ajuh, P. Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry. EMBO J. 19, 6569–6581 (2000).
pubmed: 11101529
pmcid: 305846
doi: 10.1093/emboj/19.23.6569
McCracken, S. et al. Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin. J. Biol. Chem. 280, 42227–42236 (2005).
pubmed: 16159877
doi: 10.1074/jbc.M507410200
Sharma, S., Kohlstaedt, L. A., Damianov, A., Rio, D. C. & Black, D. L. Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome. Nat. Struct. Mol. Biol. 15, 183–191 (2008).
pubmed: 18193060
pmcid: 2546704
doi: 10.1038/nsmb.1375
Rappsilber, J., Ryder, U., Lamond, A. I. & Mann, M. Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231–1245 (2002).
pubmed: 12176931
pmcid: 186633
doi: 10.1101/gr.473902
Azizian, N. G. & Li, Y. XPO1-dependent nuclear export as a target for cancer therapy. J. Hematol. Oncol. 13, 61 (2020).
pubmed: 32487143
pmcid: 7268335
doi: 10.1186/s13045-020-00903-4
Heraud-Farlow, J. E. et al. Staufen2 regulates neuronal target RNAs. Cell Rep. 5, 1511–1518 (2013).
pubmed: 24360961
doi: 10.1016/j.celrep.2013.11.039
Almasi, S. & Jasmin, B. J. The multifunctional RNA-binding protein Staufen1: an emerging regulator of oncogenesis through its various roles in key cellular events. Cell. Mol. Life Sci. 78, 7145–7160 (2021).
pubmed: 34633481
pmcid: 8629789
doi: 10.1007/s00018-021-03965-w
Yuryev, A. et al. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. Proc. Natl Acad. Sci. USA 93, 6975–6980 (1996).
pubmed: 8692929
pmcid: 38919
doi: 10.1073/pnas.93.14.6975
Tanaka, N. & Shuman, S. Structure–activity relationships in human RNA 3′-phosphate cyclase. RNA 15, 1865–1874 (2009).
pubmed: 19690099
pmcid: 2743044
doi: 10.1261/rna.1771509
Hu, X. et al. Knockdown of Trnau1ap inhibits the proliferation and migration of NIH3T3, JEG-3 and Bewo cells via the PI3K/Akt signaling pathway. Biochem. Biophys. Res. Commun. 503, 521–527 (2018).
pubmed: 29758194
doi: 10.1016/j.bbrc.2018.05.065
Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat. Methods 13, 508–514 (2016).
pubmed: 27018577
pmcid: 4887338
doi: 10.1038/nmeth.3810
Luo, Y. et al. New developments on the Encyclopedia of DNA Elements (ENCODE) data portal. Nucleic Acids Res. 48, D882–D889 (2020).
pubmed: 31713622
doi: 10.1093/nar/gkz1062
Boyle, E. A. et al. Skipper analysis of eCLIP datasets enables sensitive detection of constrained translation factor binding sites. Cell Genom. 3, 100317 (2023).
pubmed: 37388912
pmcid: 10300551
doi: 10.1016/j.xgen.2023.100317
Fairbrother, W. G., Yeh, R.-F., Sharp, P. A. & Burge, C. B. Predictive identification of exonic splicing enhancers in human genes. Science 297, 1007–1013 (2002).
pubmed: 12114529
doi: 10.1126/science.1073774
Xiao, X. et al. Splice site strength-dependent activity and genetic buffering by poly-G runs. Nat. Struct. Mol. Biol. 16, 1094–1100 (2009).
pubmed: 19749754
pmcid: 2766517
doi: 10.1038/nsmb.1661
Georgakopoulos-Soares, I. et al. Alternative splicing modulation by G-quadruplexes. Nat. Commun. 13, 2404 (2022).
pubmed: 35504902
pmcid: 9065059
doi: 10.1038/s41467-022-30071-7
Warf, M. B., Diegel, J. V., Von Hippel, P. H. & Berglund, J. A. The protein factors MBNL1 and U2AF65 bind alternative RNA structures to regulate splicing. Proc. Natl Acad. Sci. USA 106, 9203–9208 (2009).
pubmed: 19470458
pmcid: 2695092
doi: 10.1073/pnas.0900342106
Street, L. et al. Large-scale map of RNA binding protein interactomes across the mRNA life-cycle. Preprint at bioRxiv https://doi.org/10.1101/2023.06.08.544225 (2023).
Han, J. et al. Multilayered control of splicing regulatory networks by DAP3 leads to widespread alternative splicing changes in cancer. Nat. Commun. 13, 1793 (2022).
pubmed: 35379802
pmcid: 8980049
doi: 10.1038/s41467-022-29400-7
Chen, X. et al. Context-defined cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma. Nat. Commun. 14, 4259 (2023).
pubmed: 37460547
pmcid: 10352330
doi: 10.1038/s41467-023-39990-5
Uhlén, M. et al. Tissue-based map of the human proteome. Science 347, 1260419 (2015).
pubmed: 25613900
doi: 10.1126/science.1260419
Rauch, S. et al. Programmable RNA-guided RNA effector proteins built from human parts. Cell 178, 122–134 (2019).
pubmed: 31230714
pmcid: 6657360
doi: 10.1016/j.cell.2019.05.049
Zhou, Y. et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun. 10, 1523 (2019).
pubmed: 30944313
pmcid: 6447622
doi: 10.1038/s41467-019-09234-6
Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).
pubmed: 20513432
pmcid: 2898526
doi: 10.1016/j.molcel.2010.05.004
Rual, J.-F. et al. Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res. 14, 2128–2135 (2004).
pubmed: 15489335
pmcid: 528929
doi: 10.1101/gr.2973604
Blum, M. et al. The InterPro protein families and domains database: 20 years on. Nucleic Acids Res. 49, D344–D354 (2021).
pubmed: 33156333
doi: 10.1093/nar/gkaa977
The pandas development team. pandasd-dev/pandas. https://doi.org/10.5281/ZENODO.3509134 (2023).
Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17, 261–272 (2020).
pubmed: 32015543
pmcid: 7056644
doi: 10.1038/s41592-019-0686-2
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
pubmed: 22930834
pmcid: 5554542
doi: 10.1038/nmeth.2089
Durinck, S et al. biomaRt. https://doi.org/10.18129/B9.BIOC.BIOMART (2017).
Frankish, A. et al. GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res. 47, D766–D773 (2019).
pubmed: 30357393
doi: 10.1093/nar/gky955
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
pubmed: 25516281
pmcid: 4302049
doi: 10.1186/s13059-014-0550-8
Shen, S. et al. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-seq data. Proc. Natl Acad. Sci. USA 111, E5593–E5601 (2014).
pubmed: 25480548
pmcid: 4280593
doi: 10.1073/pnas.1419161111
Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification enrichment pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).
pubmed: 17703201
doi: 10.1038/nprot.2007.261
Bruderer, R. et al. Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues. Mol. Cell. Proteomics 14, 1400–1410 (2015).
pubmed: 25724911
pmcid: 4424408
doi: 10.1074/mcp.M114.044305
Wessels, H.-H. et al. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat. Biotechnol. 38, 722–727 (2020).
pubmed: 32518401
pmcid: 7294996
doi: 10.1038/s41587-020-0456-9
Guo, X. et al. Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens. Cell Genom. 1, 100001 (2021).
pubmed: 35664829
pmcid: 9164475
doi: 10.1016/j.xgen.2021.100001
Schmok, J. C. et al. Systematic identification of RNA-binding proteins and tethered domains that activate exon splicing inclusion. Gene Expression Omnibus. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE232599 (2023).