A multiprotein complex involved in chloroplast group II intron splicing

Perron, Karl; Goldschmidt-Clermont, Michel; Rochaix, Jean‐David

doi:10.1261/rna.5237804

Cited by 40 publications

(56 citation statements)

References 34 publications

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“…Previous experiments have indicated that Raa2 is associated with thylakoid membranes (25). Consequently, detergent n-dodecyl ␤-D-maltoside was added to the lysis buffer.…”

Section: Core Components Of Trans-splicing Subcomplexes I and Ii-mentioning

confidence: 99%

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns

Reifschneider¹,

Marx²,

Jacobs³

et al. 2016

Journal of Biological Chemistry

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In the chloroplast of the green alga Chlamydomonas reinhardtii, two discontinuous group II introns, psaA-i1 and psaAi2, splice in trans, and thus their excision process resembles the nuclear spliceosomal splicing pathway. Here, we address the question whether fragmentation of trans-acting RNAs is accompanied by the formation of a chloroplast spliceosome-like machinery. Using a combination of liquid chromatographymass spectrometry (LC-MS), size exclusion chromatography, and quantitative RT-PCR, we provide the first characterization of a high molecular weight ribonucleoprotein apparatus participating in psaA mRNA splicing. This supercomplex contains two subcomplexes (I and II) that are responsible for trans-splicing of either psaA-i1 or psaA-i2. We further demonstrate that both subcomplexes are associated with intron RNA, which is a prerequisite for the correct assembly of subcomplex I. This study contributes further to our view of how the eukaryotic nuclear spliceosome evolved after bacterial endosymbiosis through fragmentation of self-splicing group II introns into a dynamic, protein-rich RNP machinery.The spliceosome is a dynamic RNP machinery that participates in the excision of mRNA introns in eukaryotes. This machinery consists of five small nuclear RNAs (snRNAs; U1, U2, U4, U5, and U6) and a large number of spliceosomal proteins (1, 2). It is generally accepted that spliceosomal snRNAs were derived from ancient group II introns, which were introduced into the eukaryotic cell after endosymbiosis (3, 4). Group II introns occur frequently in bacteria and organelles of fungi, algae, and higher plants but are rare in archaea and absent from nuclear genomes. Comparable with spliceosomal introns, the splicing reaction includes two transesterification reactions, yielding spliced exons and an excised intron lariat RNA.Despite the lack of significant sequence similarities, group II introns share a common secondary structure with six helical domains (D1-D6) surrounding a central wheel. During intron excision, these domains take over specific functions, and remarkably, they show plenty of functional and structural similarities to spliceosomal snRNAs (5-7). These similarities led to the assumption that during evolution of the nuclear splicing apparatus, group II introns fragmented and degenerated to spliceosomal snRNAs (3,8,9).In contrast to bacterial group II introns, most organelle group II introns display variant forms of degeneration and fragmentation (9). For example, many of the plant group II introns have mispaired domain structures (10, 11). Moreover, group II introns are able to split into autonomous fragments due to rearrangements of organelle genomes (12, 13). Consequently, they are transcribed independently, and association of precursor RNAs by base pairing generates a catalytically active group II intron structure that is finally processed by trans-splicing. Such degenerated and fragmented group II introns along with the interplay of discrete RNAs during the splicing reaction serve to demonstrate how snRNAs...

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“…Previous experiments have indicated that Raa2 is associated with thylakoid membranes (25). Consequently, detergent n-dodecyl ␤-D-maltoside was added to the lysis buffer.…”

Section: Core Components Of Trans-splicing Subcomplexes I and Ii-mentioning

confidence: 99%

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns

Reifschneider¹,

Marx²,

Jacobs³

et al. 2016

Journal of Biological Chemistry

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“…A remarkable result of our investigation was the detection of the functionally characterized splicing factors Raa1, Raa3, and Rat2, which all are involved in splicing of the first group II intron of the psaA precursor RNA (9,10,51) (Table I). Raa1 is also involved in the splicing of second group II intron of psaA pre-mRNA (52).…”

Section: Discussionmentioning

confidence: 93%

“…Raa3, for instance, was identified in a soluble, stroma-localized ribonucleoprotein complex of about 1700 kDa that is associated with tscA RNA and psaA exon 1 precursor transcripts (9). The membrane-associated Raa1 protein, however, was identified in a complex of 670 kDa together with unidentified RNAs (10). Considering the molecular weights of these complexes, it is likely that unknown FIG.…”

Section: Discussionmentioning

confidence: 99%

“…Direct binding of Raa4 and Rab1 to tscA intron RNA was demonstrated by EMSAs. Raa1 is also involved in the splicing of psaA intron 2 and was detected in a membrane-associated complex (10). components might exist.…”

Section: Discussionmentioning

confidence: 99%

“…The unicellular green alga Chlamydomonas reinhardtii is also known to contain high molecular weight complexes containing splicing factors (9,10). In this alga, the chloroplastencoded psaA gene, which encodes a major subunit of photosystem I, is split into three independently transcribed exons.…”

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confidence: 99%

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Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components

Jacobs

Marx

Kock

et al. 2013

Molecular & Cellular Proteomics

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Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. Intron-containing genes from prokaryotic or organellar genomes carry either group I or group II introns, each of which has distinct features. The splicing mechanism of group II introns and the secondary structures of their presumed active sites were used as early arguments for the hypothesis that this class of introns represents the ancestors of eukaryotic spliceosomal introns (1, 2). It was further assumed that group II introns invaded the eukaryotic nucleus and subsequently proliferated at various genomic sites, leading to the degeneration of the catalytic intron structure into small nuclear RNAs (snRNAs).1 This assumption was supported by the observation of naturally occurring variants of group II introns that are split into two or more pieces (3), reminiscent of eukaryotic spliceosomal RNA (1). Group II intron RNAs are characterized by six conserved domains, and tertiary interactions among these domains generate the compact native and catalytic complex. Some of these group II intron domains have been shown to act in trans on the splicing of other introns that lack the corresponding domain (4). In vivo, various RNA-binding proteins promote the formation of catalytically active intron RNA. In contrast to the nuclear spliceosome, which acts generally on a broad range of nuclear-encoded pre-mRNAs, proteins involved in organellar intron splicing seem to more efficiently stabilize the active three-dimensional RNA structure in vivo. Several splicing factors in higher plants, such as the chloroplast RNA-splicing and ribosome maturation (CRM) domain protein CRS1, as well as the pentatricopeptide repeat proteins OTP51 and PPR4, have been reported to be involved in the splicing of single transcripts (5, 6). Nonetheless, there are splicing factors that carry out functions on a broad range of transcripts, including CRS2 and its associated proteins CAF1 and CAF2, and WTF1, a splicing factor containing a plant organelle RNA-recognition domain (5, 6). Sedimentation and co-fractionation experiments in, for example, maize have demonstrated that these proteins are part of large multiprotein and ribonucleoprotein complexes with their cognate RNAs (5, 7). I...

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Strategies for the Investigation of Protein–Protein Interactions in Plants

Braun

Schmitz

Plant Proteomics

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A multiprotein complex involved in chloroplast group II intron splicing

Cited by 40 publications

References 34 publications

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns

A Ribonucleoprotein Supercomplex Involved in trans-Splicing of Organelle Group II Introns

Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components

Strategies for the Investigation of Protein–Protein Interactions in Plants

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