Mutational analysis of the U12-dependent branch site consensus sequence

Brock, Jay E; Dietrich, Rosemary C.; Padgett, Richard A.

doi:10.1261/rna.1189008

Cited by 8 publications

(16 citation statements)

References 44 publications

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“…The U11 GG6/7CC, U6atac GG14/15CC and U12 GA23/24CU expression plasmids have been described previously 10 11 12 14 23 24 36 . Second site mutations were introduced in U6atac GG14/15CC and U12 GA23/24CU snRNAs to construct swapped SL snRNA plasmids and U6atac distal 3′ SL mutant plasmids.…”

Section: Methodsmentioning

confidence: 99%

U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

Singh

Sikand

Conrad

et al. 2016

Sci Rep

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Formation of catalytic core of the U12-dependent spliceosome involves U6atac and U12 interaction with the 5′ splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3′ stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3′ stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3′ stem-loop. Furthermore, we tested the compatibility of the U6atac 3′ end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the minor spliceosome are highly plastic as compared to the major spliceosome.

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Section: Methodsmentioning

confidence: 99%

U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

Singh

Sikand

Conrad

et al. 2016

Sci Rep

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“…The starting U12 snRNA expression plasmid has been described previously ( 8 ). This construct contains mutations GA23/24CU in the branch site interacting region to restore complementary base pairing to UC84/85AG branch site region of P120 U12-dependent intron F. For this study, second site mutations were introduced in U12 snRNA in the background of GA23/24CU.…”

Section: Methodsmentioning

confidence: 99%

“…U2 snRNA binds the branch site sequences by canonical base pairing interactions, which are necessary for U2-dependent intron splicing ( 7 ). Similarly, U12 snRNA binds to the branch site of a U12-dependent intron by RNA-RNA base pairing essential for splicing ( 3 , 8 ). Besides binding to the branch site sequence, U2 snRNA also base pairs with U6 snRNA to form an intermolecular helix required for U2-dependent splicing ( 9–12 ).…”

Section: Introductionmentioning

confidence: 99%

Functionally important structural elements of U12 snRNA

Sikand

Shukla

2011

Nucleic Acids Research

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U12 snRNA is analogous to U2 snRNA of the U2-dependent spliceosome and is essential for the splicing of U12-dependent introns in metazoan cells. The essential region of U12 snRNA, which base pairs to the branch site of minor class introns is well characterized. However, other regions which are outside of the branch site base pairing region are not yet characterized and the requirement of these structures in U12-dependent splicing is not clear. U12 snRNA is predicted to form an intricate secondary structure containing several stem–loops and single-stranded regions. Using a previously characterized branch site genetic suppression assay, we generated second-site mutations in the suppressor U12 snRNA to investigate the in vivo requirement of structural elements in U12-dependent splicing. Our results show that stem–loop IIa is essential and required for in vivo splicing. Interestingly, an evolutionarily conserved stem–loop IIb is dispensable for splicing. We also show that stem–loop III, which binds to a p65 RNA binding protein of the U11-U12 di.snRNP complex, is essential for in vivo splicing. The data validate the existence of proposed stem–loops of U12 snRNA and provide experimental support for individual secondary structures.

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“…Naturally occurring U12‐type introns mostly belong to either the AT–AC or GT–AG subtypes. A few natural examples of U12‐type introns with other combinations of terminal residues have been reported, and non‐canonical termini have also been shown to support U12‐dependent splicing experimentally, although often with reduced efficiency 11–15. However, combinations of the main subtypes (i.e., AT–AG or GT–AC) appear to be disfavored.…”

Section: U12‐type Intron Sequences: Definition and Characteristicsmentioning

confidence: 99%

The significant other: splicing by the minor spliceosome

et al. 2012

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The removal of non-coding sequences, introns, from the mRNA precursors is an essential step in eukaryotic gene expression. U12-type introns are a minor subgroup of introns, distinct from the major or U2-type introns. U12-type introns are present in most eukaryotes but only account for less than 0.5% of all introns in any given genome. They are processed by a specific U12-dependent spliceosome, which is similar to, but distinct from, the major spliceosome. U12-type introns are spliced somewhat less efficiently than the major introns, and it is believed that this limits the expression of the genes containing such introns. Recent findings on the role of U12-dependent splicing in development and human disease have shown that it can also affect multiple cellular processes not directly related to the functions of the host genes of U12-type introns. At the same time, advances in understanding the regulation and phylogenetic distribution of the minor spliceosome are starting to shed light on how the U12-type introns and the minor spliceosome may have evolved. © 2012 John Wiley & Sons, Ltd.

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Mutational analysis of the U12-dependent branch site consensus sequence

Cited by 8 publications

References 44 publications

U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

Functionally important structural elements of U12 snRNA

The significant other: splicing by the minor spliceosome

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