Abstract
Informational suppression is a method to map specific RNA:RNA interactions by taking advantage of the rules of base complementarity. First, a predicted Watson-Crick base pair is broken by single-nucleotide substitution which disrupts the RNA’s structure and/or function. Second, the base pair is restored by mutating the opposing nucleotide, thereby rescuing structure and/or function. This method applies to RNP:RNA interactions such as 5′ splice-site (5′ss) base-pairing to the 5′ end of U1 small nuclear RNA as part of a small nuclear RNP. Our protocol aims to determine the 5′ss:U1 base-pairing register for natural 5′ss, because for distinct 5′ss sequences the nucleotides on each strand can be aligned differently. This methodology includes cloning of a wild-type splicing minigene and introduction of 5′ss variants by PCR mutagenesis. A U1-expression plasmid is mutated to construct “suppressor U1” snRNAs with restored base-pairing to mutant 5′ss in different registers. Cells are transfected with combinations of minigenes and suppressor U1s, and the splicing patterns are analyzed by reverse transcription and semiquantitative PCR, followed by gel electrophoresis. The identity of suppressor U1s that rescue splicing for specific mutations indicates the register used in that 5′ss. We also provide tips to adapt this protocol to other minigenes or registers.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Woolford JL Jr, Baserga SJ (2013) Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195:643–681
Fischer U, Englbrecht C, Chari A (2011) Biogenesis of spliceosomal small nuclear ribonucleoproteins. Wiley Interdiscip Rev RNA 2:718–731
Brow DA (2002) Allosteric cascade of spliceosome activation. Annu Rev Genet 36:333–360
Wahl MC, Will CL, Lührmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136:701–718
Roca X, Krainer AR, Eperon IC (2013) Pick one, but be quick: 5′ splice sites and the problems of too many choices. Genes Dev 27:129–144
Sheth N, Roca X, Hastings ML et al (2006) Comprehensive splice-site analysis using comparative genomics. Nucleic Acids Res 34:3955–3967
Heinrichs V, Bach M, Winkelmann G et al (1990) U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5′ splice site. Science 247:69–72
Kondo Y, Oubridge C, van Roon AM et al (2015) Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition. Elife 4
Murgola EJ (1985) tRNA, suppression and the code. Annu Rev Genet 19:57–80
Prelich G (1999) Suppression mechanisms: themes from variations. Trends Genet 15:261–266
Mount SM, Anderson P (2000) Expanding the definition of informational suppression. Trends Genet 16:157
Zhuang Y, Weiner AM (1986) A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation. Cell 46:827–835
Séraphin B, Kretzner L, Rosbash MH (1988) A U1 snRNA:premRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. EMBO J 7:2533–2538
Siliciano PG, Guthrie C (1988) 5′ splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev 2:1258–1267
Carmel I, Tal S, Vig I et al (2004) Comparative analysis detects dependencies among the 5′ splice-site positions. RNA 10:828–840
Cohen JB, Snow JE, Spencer SD et al (1994) Suppression of mammalian 5′ splice-site defects by U1 small nuclear RNAs from a distance. Proc Natl Acad Sci U S A 91:10470–10474
Lo PC, Roy D, Mount SM (1994) Suppressor U1 snRNAs in Drosophila. Genetics 138:365–378
Pinotti M, Bernardi F, Dal Mas A et al (2011) RNA-based therapeutic approaches for coagulation factor deficiencies. J Thromb Haemost 9:2143–2152
Kandels-Lewis S, Séraphin B (1993) Involvement of U6 snRNA in 5′ splice site selection. Science 262:2035–2039
Lesser CF, Guthrie C (1993) Mutations in U6 snRNA that alter splice site specificity: Implications for the active site. Science 6:1982–1988
Wassarman DA, Steitz JA (1992) Interactions of small nuclear RNA’s with precursor messenger RNA during in vitro splicing. Science 257:1918–1925
Hwang DY, Cohen JB (1996) U1 snRNA promotes the selection of nearby 5′ splice sites by U6 snRNA in mammalian cells. Genes Dev 10:338–350
Roca X, Krainer AR (2009) Recognition of atypical 5′ splice sites by shifted base-pairing to U1 snRNA. Nat Struct Mol Biol 16:176–182
Roca X, Akerman M, Gaus H et al (2012) Widespread recognition of 5′ splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides. Genes Dev 26:1098–1109
Murphy JT, Skuzeski JT, Lund E et al (1987) Functional elements of the human U1 RNA promoter. Identification of five separate regions required for efficient transcription and template competition. J Biol Chem 262:1795–1803
Markham NR, Zuker M (2008) UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol 453:3–31
Flicek P, Amode MR, Barrell D et al (2014) Ensembl 2014. Nucleic Acids Res 42:D749–D755
Lorson CL, Hahnen E, Androphy EJ et al (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci U S A 96:6307–6311
Will CL, Rümpler S, Klein Gunnewiek J et al (1996) In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes. Nucleic Acids Res 24:4614–4623
Roca X, Olson AJ, Rao AR et al (2008) Features of 5′-splice-site efficiency derived from disease-causing mutations and comparative genomics. Genome Res 18:77–87
Baserga SJ, Steitz JA (1993) In: Gesteland RF, Atkins JF (eds) The RNA world. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p 359–381
Sharma S, Wongpalee SP, Vashisht A et al (2014) Stem-loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly. Genes Dev 28:2518–2531
Cartegni L, Hastings ML, Calarco JA et al (2006) Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2. Am J Hum Genet 78:63–77
Acknowledgments
XR acknowledges funding from Academic Research Fund Tier 1 grant (RG 20/11) from Singapore’s Ministry of Education, as well as a Startup Grant from School of Biological Sciences at Nanyang Technological University, Singapore. The authors thank Ms Jia Xin Jessie Ho for details of some protocols.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Tan, J., Roca, X. (2016). Informational Suppression to Probe RNA:RNA Interactions in the Context of Ribonucleoproteins: U1 and 5′ Splice-Site Base-Pairing. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 1421. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3591-8_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3591-8_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3589-5
Online ISBN: 978-1-4939-3591-8
eBook Packages: Springer Protocols