Molecular consequences of truncations of the first exon for<i>in vitro</i>splicing of yeast actin pre-mRNA

Duchêne, Michael; Löw, Angela; Schweizer, Andrea; Domdey, Horst

doi:10.1093/nar/16.15.7233

Cited by 12 publications

(8 citation statements)

References 22 publications

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“…Yet, most of these 5Ј exon interactions are clearly dispensable for splicing in vitro. In both yeast and human splicing extracts, no decrease in first-step splicing efficiency is observed when the 5Ј exon is truncated to 2 nt, and even a 1-nt exon can support some lariat formation (Duchene et al 1988;Hertel and Maniatis 1999). In our hands, an AdML substrate with a 6-nt 5Ј exon underwent efficient lariat formation, but was significantly impaired for exon ligation (data not shown).…”

Section: Protection Of the 5ј Exon Intermediate Is Extensivementioning

confidence: 57%

“…A 14-nt 5Ј exon did undergo exon ligation, but not as efficiently as longer sequences (data not shown). Similarly, efficient exon ligation in S. cerevisiae extracts requires the 5Ј exon to be somewhere between 6 and 12 nt (Duchene et al 1988). Thus, at least in vitro, truncation of the 5Ј exon is of little or no consequence for the first step of splicing, but there is a minimum 5Ј exon length requirement for the second step.…”

Section: Protection Of the 5ј Exon Intermediate Is Extensivementioning

confidence: 99%

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5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Reichert

Hir²,

Jurica³

et al. 2002

Genes Dev.

126

137

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A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5 exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5 exon intermediate. Throughout their lifetimes in vivo, mRNAs exist as mRNA-protein particles (mRNPs). The associated proteins control every aspect of mRNA metabolism, from subcellular transport to translational efficiency to rate of decay. Exactly which proteins associate with a particular mRNA depends on its sequence, its subcellular localization, and its synthetic history. Furthermore, the complement of mRNP proteins evolves as the mRNA moves to different locations and is acted on by such processes as nuclear export and translation. Many proteins join the mRNP in the nucleus and then accompany it to the cytoplasm, where they influence subsequent mRNA metabolism ( These splicing-specific mRNP proteins constitute the exon junction complex (EJC), which associates with spliced mRNAs in a sequence-independent manner a fixed distance upstream of exon-exon junctions (Le Hir et al. 2000b). Using coimmunoprecipitation strategies, we and others recently reported that the EJC assembled in HeLa nuclear extract contains numerous epitopes, including those recognized by antibodies against REF/Aly, Y14, SRm160, DEK, RNPS1, UAP56, and Magoh (Blencowe et al. 1998;Mayeda et al. 1999;Kataoka et al. 2000Kataoka et al. , 2001Le Hir et al. 2000a,b, 2001bMcGarvey et al. 2000;Zhou et al. 2000;Gatfield et al. 2001;Luo et al. 2001). When assembled in vivo, the EJC is additionally precipitated by antibodies against Upf2, Upf3, and the TAP:p15 heterodimer (Kim et al. 2001b;Le Hir et al. 2001a;Lykke-Andersen et al. 2001). Finally, REF/Aly, Y14, SRm160, RNPS1, Upf2, Upf3, and TAP were all present in mRNPs immunopurified from the nuclear fraction of COS cells with antibodies against a subunit of the nuclear cap binding complex (Lejeune et al. 2002).The known EJC components function at multiple levels of mRNA metabolism. SRm160 and RNPS1 were originally characterized as activators of pre-mRNA splicing (Blencowe et al. 1998;Mayeda et al. 1999

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Section: Protection Of the 5ј Exon Intermediate Is Extensivementioning

confidence: 57%

Section: Protection Of the 5ј Exon Intermediate Is Extensivementioning

confidence: 99%

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Reichert

Hir²,

Jurica³

et al. 2002

Genes Dev.

126

137

View full text Add to dashboard Cite

show abstract

“…To investigate the function(s) of the EJC apart from any other possible effects of pre‐mRNA splicing on mRNA metabolism, we wanted to generate mRNAs that had been produced by splicing but did not carry the complex. It was previously reported that pre‐mRNAs having a 5′ exon as short as 12 nt can support both steps of splicing in vitro (Duchene et al ., 1988; Chanfreau et al ., 1999). Since the EJC is deposited on spliced mRNAs >20 nt upstream of the 3′ end of the 5′ exon, we hypothesized that spliced mRNAs with 5′ exons <20 nt might not carry the complex.…”

Section: Resultsmentioning

confidence: 99%

The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay

2001

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We recently reported that spliceosomes alter messenger ribonucleoprotein particle (mRNP) composition by depositing several proteins 20±24 nucleotides upstream of mRNA exon±exon junctions. When assembled in vitro, this so-called`exon±exon junction complex' (EJC) contains at least ®ve proteins: SRm160, DEK, RNPS1, Y14 and REF. To better investigate its functional attributes, we now describe a method for generating spliced mRNAs both in vitro and in vivo that either do or do not carry the EJC. Analysis of these mRNAs in Xenopus laevis oocytes revealed that this complex is the species responsible for enhancing nucleocytoplasmic export of spliced mRNAs. It does so by providing a strong binding site for the mRNA export factors REF and TAP/p15. Moreover, by serving as an anchoring point for the factors Upf2 and Upf3, the EJC provides a direct link between splicing and nonsense-mediated mRNA decay. Finally, we show that the composition of the EJC is dynamic in vivo and is subject to signi®cant evolution upon mRNA export to the cytoplasm.

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“…Neither sequence substitution caused a significant change in trans-splicing efficiency ( Figure 3B). Domdey and coworkers (Duchenne et al, 1988) showed in a yeast system that constructs containing first exons of 1, 2, 3 or 5 nucleotides participated only in the first step of splicing and did not participate in the second step. This mutation caused a modest reduction (30%) in utilization of the SL RNA, but as with the C25C26A27A28 mutation in stem I, the reduction in utilization was directly correlated with inefficient assembly of the shortened SL RNA (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Intramolecular base pairing between the nematode spliced leader and its 5′ splice site is not essential for trans-splicing in vitro.

et al. 1991

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The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans‐splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans‐splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans‐splice donor site did not affect the ability of the SL RNA to function in trans‐splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the ‘intron’ portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans‐spliceosome.

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Molecular consequences of truncations of the first exon forin vitrosplicing of yeast actin pre-mRNA

Cited by 12 publications

References 22 publications

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay

Intramolecular base pairing between the nematode spliced leader and its 5′ splice site is not essential for trans-splicing in vitro.

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