Mammalian In Vitro Splicing Assays

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity.

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In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3428-3437.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3428-3437

Author(s):

K M Lang ◽

R A Spritz

Keyword(s):

Temporal Order ◽

Exon Skipping ◽

Beta Globin ◽

Splice Site Selection ◽

Interleukin 3 ◽

Intervening Sequences ◽

In Vitro Splicing ◽

Lag Times ◽

Alpha Lactalbumin

We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.

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An in vitro splicing assay reveals the pathogenicity of a novel intronic variant in ATP6V0A4 for autosomal recessive distal renal tubular acidosis

BMC Nephrology ◽

10.1186/s12882-017-0774-4 ◽

2017 ◽

Vol 18 (1) ◽

Cited By ~ 5

Author(s):

Tomohiko Yamamura ◽

Kandai Nozu ◽

Yuya Miyoshi ◽

Keita Nakanishi ◽

Junya Fujimura ◽

...

Keyword(s):

Renal Tubular Acidosis ◽

Autosomal Recessive ◽

Distal Renal Tubular Acidosis ◽

In Vitro Splicing ◽

Renal Tubular

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Mutation of the AAUAAA polyadenylation signal depresses in vitro splicing of proximal but not distal introns.

Genes & Development ◽

10.1101/gad.5.11.2086 ◽

1991 ◽

Vol 5 (11) ◽

pp. 2086-2095 ◽

Cited By ~ 97

Author(s):

M Niwa ◽

S M Berget

Keyword(s):

Polyadenylation Signal ◽

In Vitro Splicing

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Polypurine sequences within a downstream exon function as a splicing enhancer.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1347 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1347-1354 ◽

Cited By ~ 120

Author(s):

K Tanaka ◽

A Watakabe ◽

Y Shimura

Keyword(s):

Recognition Sequence ◽

Splice Site Selection ◽

Sequence Element ◽

Nuclear Extracts ◽

Gene Functions ◽

Mouse Immunoglobulin ◽

In Vitro Splicing ◽

Immunoglobulin Mu ◽

Splicing Enhancer

We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.

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