SHAPE Profiling to Probe Group II Intron Conformational Dynamics During Splicing.
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ABSTRACT:
SUBMITTER: Wiryaman T
PROVIDER: S-EPMC7510887 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5'-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.
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Project description:Group II introns are large catalytic RNAs (ribozymes) which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA) in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has been retained in the mtDNA of various angiosperms: the matR gene encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4). Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4), which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory splicing cofactors.
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Project description:The autocatalytic group II intron ai5? from Saccharomyces cerevisiae self-splices under high-salt conditions in vitro, but requires the assistance of the DEAD-box protein Mss116 in vivo and under near-physiological conditions in vitro. Here, we show that Mss116 influences the folding mechanism in several ways. By comparing intron precursor RNAs with long (?300 nt) and short (?20 nt) exons, we observe that long exon sequences are a major obstacle for self-splicing in vitro. Kinetic analysis indicates that Mss116 not only mitigates the inhibitory effects of long exons, but also assists folding of the intron core. Moreover, a mutation in conserved Motif III that impairs unwinding activity (SAT ? AAA) only affects the construct with long exons, suggesting helicase unwinding during exon unfolding, but not in intron folding. Strong parallels between Mss116 and the related protein Cyt-19 from Neurospora crassa suggest that these proteins form a subclass of DEAD-box proteins that possess a versatile repertoire of diverse activities for resolving the folding problems of large RNAs.
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Project description:Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report 14 crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, and they adopt a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome.
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Project description:While the major architectural features and active-site components of group II introns have been known for almost a decade, information on the individual stages of splicing has been lacking. Recent advances in crystallography and cryo-electron microscopy (cryo-EM) have provided major new insights into the structure of intact lariat introns. Conformational changes that mediate the steps of splicing and retrotransposition are being elucidated, revealing the dynamic, highly coordinated motions that are required for group II intron activity. Finally, these ribozymes can now be viewed in their larger, more natural context as components of holoenzymes that include encoded maturase proteins. These studies expand our understanding of group II intron structural diversity and evolution, while setting the stage for rigorous mechanistic analysis of RNA splicing machines.
| S-EPMC5492998 | biostudies-literature
Project description:Group II introns, the putative progenitors of spliceosomal introns and retrotransposons, are ribozymes that are capable of self-splicing and DNA invasion. In the cell, group II introns form ribonucleoprotein (RNP) complexes with an intron-encoded protein, which is essential to folding, splicing and retromobility of the intron. To understand the structural accommodations underlying splicing, in preparation for retromobility, we probed the endogenously expressed Lactococcus lactis Ll.LtrB group II intron RNP using SHAPE. The results, which are consistent in vivo and in vitro, provide insights into the dynamics of the intron RNP as well as RNA-RNA and RNA-protein interactions. By comparing the excised intron RNP with mutant RNPs in the precursor state, confined SHAPE profile differences were observed, indicative of rearrangements at the active site as well as disengagement at the functional RNA-protein interface in transition between the two states. The exon-binding sequences in the intron RNA, which interact with the 5' exon and the target DNA, show increased flexibility after splicing. In contrast, stability of major tertiary and protein interactions maintains the scaffold of the RNA through the splicing transition, while the active site is realigned in preparation for retromobility.
| S-EPMC6144810 | biostudies-literature
Project description:The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of Ll.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (10(7)-fold detection range), we demonstrate that Ll.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for Ll.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented Ll.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells.
| S-EPMC1874635 | biostudies-literature
Project description:The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3' exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3' splice site within the catalytic core of the group II intron. The 3'-OH of the 5' exon is positioned in close proximity to the 3' splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having different configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3' splice site capture.
| S-EPMC6224600 | biostudies-literature
Project description:The thermostable Geobacillus stearothermophilus GsI-IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI-IIC. We found that GsI-IIC, like other group IIC introns, retrohomes into sites having a 5'-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7-19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5'-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5'-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI-IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream.
| S-EPMC6082379 | biostudies-literature
Project description:The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.
| S-EPMC1087544 | biostudies-literature