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Group II introns: versatile ribozymes and retroelements

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Group II introns are catalytic RNAs (ribozymes) and retroelements found in the genomes of bacteria, archaebacteria, and organelles of some eukaryotes. The prototypical retroelement form consists of a structurally conserved RNA and a multidomain reverse transcriptase protein, which interact with each other to mediate splicing and mobility reactions. A wealth of biochemical, cross‐linking, and X‐ray crystal structure studies have helped to reveal how the two components cooperate to carry out the splicing and mobility reactions. In addition to the standard retroelement form, group II introns have evolved into derivative forms by either losing specific splicing or mobility characteristics, or becoming functionally specialized. Of particular interest are the eukaryotic derivatives—the spliceosome, spliceosomal introns, and non‐LTR retroelements—which together make up approximately half of the human genome. On a practical level, the properties of group II introns have been exploited to develop group II intron‐based biotechnological tools. WIREs RNA 2016, 7:341–355. doi: 10.1002/wrna.1339 This article is categorized under: RNA Processing > Splicing Mechanisms RNA-Based Catalysis > RNA Catalysis in Splicing and Translation
Splicing reactions. (a) Standard splicing reaction, proceeding through two transesterifications and a lariat intermediate, to yield spliced exons and intron lariat. The schematic shows the splicing reaction with facilitation by the intron‐encoded protein (IEP), although the identical reaction occurs without the IEP under self‐splicing conditions in vitro. (b) Splicing through hydrolysis. The first step of splicing is initiated by a water nucleophile rather than the 2′ OH of an adenosine. This results in a linear intermediate, and the intron being released in a linear form rather than a lariat.
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Genomic structure and RNA secondary structure of a group II intron. The schematics in both panels correspond to the IIA intron Ll.LtrB of Lactococcus lactis. (a) Genomic structure. The intron consists of a ribozyme component (red) and protein component (different shades of blue). The RNA component has six structural domains (bracketed below), with domain 4 split into two parts (4a, 4b). The intron‐encoded protein consists of RT motifs 0–7 (palm and finger domains of the RT), domain X (thumb domain of the RT and required for maturase activity), a DNA‐binding domain D, and an endonuclease domain En, which is lacking from some introns. The intron is nested between two exons, E1 and E2 (green). (b) RNA secondary structure. The ribozyme's secondary structure is in red, beginning with the 5′ boundary motif GUGYG and ending with AY. The intron‐encoded protein (IEP)’s open reading frame (ORF) is located within the loop of D4 (shades of blue), and the IEP‐binding site is indicated by dotted red lines. The 5′ and 3′ exons are in green. Tertiary interactions within the RNA structure are denoted by Greek lettering (e.g., α–α′). For IIA introns, the pairings between exons and introns occur through IBS1–EBS1, IBS2–EBS2, and δ–δ′. See Figures and for pairing motifs of IIB and IIC introns. The interactions μ–μ′, π–π′, and ρ–ρ′ are not shown because they have not been defined for IIA introns; however, they are depicted in Figure for IIB introns.
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Comparison of the major similarities between group II introns and the snRNAs of the spliceosome. (a) Schematic of group II intron motifs having putative parallels in the spliceosome. (b) Schematic of RNA pairings present in activated spliceosomes that resemble group II intron structures. The D5 active site of group II introns is recapitulated in the spliceosome by the U6 internal stem‐loop (ISL) and the U2–U6 snRNA pairing, including an AGC sequence, a two‐nucleotide bulge, and triple base pairs with the catalytic triad. The D6 branch site of group II introns is formed analogously in the spliceosome by the U2 snRNA pairing with the branch‐site sequence near the 3′ end of the intron. A pairing between the ACAGAGA sequence of the U6 snRNA and the 5′ end of the intron is proposed to be analogous to the ε–ε′ pairing. Finally, pairings between the exons and U5 snRNA resemble the EBS1–EBS1 pairings of all group II introns and the δ−δ′ pairing of IIA introns.
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The target‐primed reverse transcription (TPRT) mechanism of retrohoming. The reaction is initiated by a ribonucleoprotein (RNP) consisting of intron lariat and the intron‐encoded protein (IEP; the end product of splicing). The RNP recognizes the dsDNA target and unwinds the strands, allowing the intron to reverse splice into the top strand of the DNA. The bottom strand is cleaved by the En domain of the IEP, and the cleaved DNA is the primer for reverse transcription of the intron RNA. Repair activities present in the host cell convert the inserted intron to dsDNA.
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Tertiary structure. (a) X‐ray crystal structure of the IIC intron of Oceanobacillus iheyensis (PDB ID 3IGI), with domains color‐coded according to the secondary structure shown at the bottom. Only the backbone is shown except for D5, the exons, and the positions that pair with the exons. D5 is in red with the AC bulge nucleotides in solid blue and the catalytic triad CGC nucleotides in solid purple. The two catalytic metal ions are in yellow. Bound exons are light blue. The crystallized intron construct corresponds to the structure at the bottom, with tertiary interactions denoted with light blue shadings. Dotted lines indicate portions of the wild‐type intron that were deleted from the crystallized construct (D2, D3, distal portion of D6) or were not present in the crystallized structure (entirety of D6). A close‐up of the active site is shown in the middle. (b) X‐ray crystal structure of the IIB intron P.li.LSUI2 of the alga Pylaiella littoralis mitochondrion, color coded according to the secondary structure shown below. The 2′–5′ branch connection between the bulged A and 5′ end of the intron is shown in pink; however, only the sugar and phosphate are shown because the base was not visible in the crystal structure. The break in domain 1 in the secondary and tertiary structures is due to unresolved nucleotides in the crystal structure. A close‐up of the active site is shown in the middle panel of the figure.
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Mechanisms of exon recognition for IIA, IIB, and IIC introns. Base‐pairing interactions used by IIA, IIB, and IIC introns to bind the exons at the active site. For simplicity the intron sequence is omitted and the exon junction is indicated by a vertical black line. EBS, exon‐binding site; IBS, intron‐binding site. (a) For IIA introns, the 5′ exon is recognized by IBS1–EBS1 and IBS2–EBS2 interactions, and the 3′ exon by the δ–δ′ interaction. (b) For IIB introns, the 5′ exon is recognized by IBS1–EBS1 and IBS2–EBS2 interactions, and the 3′ exon by the IBS3–EBS3 interaction. (c) For IIC introns, the 5′ exon is recognized by a shortened IBS1–EBS1 interaction, and the 3′ exon by the IBS3–EBS3 interaction.
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Variations in the splicing reaction. (a) 5′ Exon cleavage reaction. A minimal ribozyme construct that consists of D1 and D5 cleaves the 5′ exon from the intron through a hydrolysis reaction. (b) Spliced‐exon reopening (SER) reaction. Spliced intron lariat attacks ligated exons in a reverse‐splicing‐like reaction, leading to cleavage of the exons. (c) Trans‐splicing. A group II intron is transcribed in two pieces, which reassociate to splice together exons in trans from separate transcripts. (d) Model for circle formation. A molecule of 5′ exon/intron is produced through the attack of 5′ exon (produced by SER) on unspliced intron. The 5′ exon/intron molecule is circularized by the attack of its 3′ end on it 5′ exon–intron junction.
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