Tissue-Specific Splicing in Vivo of the β-Tropomyosin Gene: Dependence on an RNA Secondary Structure

Libri, Domenico; Piseri, Anna; Fiszman, Marc

doi:10.1126/science.2063196

Cited by 148 publications

(95 citation statements)

References 20 publications

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“…First, the stem-loop alone might inhibit the use of this 39 splice site; second, the addition of MBNL might further stabilize this stem-loop and increase the block at this 39 splice site. Previously, stem-loop formation has been shown to reduce the use of 59 and 39 splice sites (Watakabe et al 1989;Libri et al 1991), but this is the first example to our knowledge in which the stem-loop is specifically recognized by a factor that regulates splicing. Therefore MBNL may be a member of a new second class of splicing regulators utilizing secondary structure, while other splicing regulators have previously been shown to recognize single-stranded motifs, such as the SR splicing factors (for review, see Blencowe et al 1999;Singh and Valcarcel 2005).…”

Section: Regulated Splicing By Mbnl Requires a Stem-loop Containing Amentioning

confidence: 99%

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T

Warf

Berglund²

2007

RNA

157

278

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Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. We demonstrate that MBNL can bind short structured CUG and CCUG repeats with high affinity and specificity. Only 6 base pairs are necessary for MBNL binding: two pyrimidine mismatches and four guanosinecytosine base pairs in a stem. MBNL also has a preference for pyrimidine mismatches, but many other mismatches are tolerated with decreased affinity. We also demonstrate that MBNL binds the helical region of a stem-loop in the endogenous pre-mRNA target, the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. In vivo splicing results indicate that MBNL-regulated splicing is dependent upon the formation of stem-loops recognized by MBNL. These results suggest that MBNL may bind all of its RNA substrates, both normal and pathogenic, as structured stem-loops containing pyrimidine mismatches.

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Section: Regulated Splicing By Mbnl Requires a Stem-loop Containing Amentioning

confidence: 99%

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T

Warf

Berglund²

2007

RNA

157

278

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“…A secondary structure was shown in chicken ␤-TM pre-mRNA (D'Orval et al 1991;Libri et al 1991), and stem I (see Fig. 1C) plays a key role in the stabilization of this structure.…”

Section: An In Vitro System To Study the Ex-1 Mutationmentioning

confidence: 99%

Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta -tropomyosin gene

Chen¹,

Kobayashi²,

Helfman³

1999

Genes & Development

186

145

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In the rat ␤-tropomyosin (␤-TM) gene, exons 6 and 7 are spliced alternatively in a mutually exclusive manner. Exon 6 is included in mRNA encoding nonmuscle TM-1, whereas exon 7 is used in mRNA encoding skeletal muscle ␤-TM. Previously, we demonstrated that a six nucleotide mutation at the 5 end of exon 7, designated as ex-1, activated exon 7 splicing in nonmuscle cells. In this study, we show that the activating effect of this mutation is not the result of creating an exonic splicing enhancer (ESE) or disrupting a putative secondary structure. The sequence in exon 7 acts as a bona fide exonic splicing silencer (ESS), which is bound specifically by a trans-acting factor. Isolation and peptide sequencing reveal that this factor is hnRNP H, a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Binding of hnRNP H correlates with the ESS activity. Furthermore, addition of antibodies that specifically recognizes hnRNP H to the splicing reactions or partial depletion of hnRNP H from nuclear extract activates exon 7 splicing in vitro and this effect can be reversed by addition of purified recombinant hnRNP H. These results indicate that hnRNP H participates in exclusion of exon 7 in nonmuscle cells. The involvement of hnRNP H in the activity of an ESS may represent a prototype for the regulation of tissue-and developmental-specific alternative splicing.

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“…The second involves the presence of mRNA secondary structures. The formation of hairpin loops, for instance, could make the splice site sterically inaccessible for spliceosome formation, as has been described for β-tropomyosin alternative processing [33][34][35][36][37]. Sequences surrounding the alternatively included muscle-specific exon 7 of β-tropomyosin were shown to be involved in the formation of a stable secondary structure.…”

Section: Discussionmentioning

confidence: 99%

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript

Bosch

Mertens

Gijsbers

et al. 1997

Biochemical Journal

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Expression of the muscle-specific 2a isoform of the sarco\ endoplasmic reticulum Ca# + -ATPase (SERCA2) requires activation of an inefficient optional splice process at the 3h end of the primary gene transcript. The sequence elements required for this regulated splice event were studied by modifying a minigene containing the 3h end of the SERCA2 gene. An important requirement appears to be a strong muscle-specific acceptor site, as replacing it by a weak one prevented the induction of muscletype splicing during myogenic differentiation. The induction of muscle-type splicing did not depend on positive cis-active sequences in the muscle-specific exon. On the other hand,

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Tissue-Specific Splicing in Vivo of the β-Tropomyosin Gene: Dependence on an RNA Secondary Structure

Cited by 148 publications

References 20 publications

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T

Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat beta -tropomyosin gene

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript

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