Group II intron domain 5 facilitates a trans-splicing reaction.

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.

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Methylation of rRNA as a host defense against rampant group II intron retrotransposition

Mobile DNA ◽

10.1186/s13100-021-00237-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Justin M. Waldern ◽

Dorie Smith ◽

Carol Lyn Piazza ◽

E. Jake Bailey ◽

Nicholas J. Schiraldi ◽

...

Keyword(s):

Insertional Mutagenesis ◽

Fold Increase ◽

Group Ii Intron ◽

In Vivo Experiments ◽

Cellular Factors ◽

Group Ii ◽

Self Splicing ◽

Native Host

Abstract Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

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Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4479 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4479-4488 ◽

Cited By ~ 37

Author(s):

S C Boulanger ◽

S M Belcher ◽

U Schmidt ◽

S D Dib-Hajj ◽

T Schmidt ◽

...

Keyword(s):

Point Mutations ◽

Group Ii Intron ◽

The Other ◽

Base Pairs ◽

Saccharomyces Cerevisiae Mitochondria ◽

Small Nuclear Rnas ◽

Group Ii ◽

Self Splicing

Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing. Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant. We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria. Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4. Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype. Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function. At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing. A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5.

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Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5896 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5896-5904 ◽

Cited By ~ 14

Author(s):

S C Boulanger ◽

P H Faix ◽

H Yang ◽

J Zhuo ◽

J S Franzen ◽

...

Keyword(s):

Splice Junction ◽

Type A ◽

Splice Site Selection ◽

Length Changes ◽

Group Ii ◽

Reduced Rate ◽

Self Splicing ◽

Yeast Mitochondrial Dna

Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme. Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately. The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted. Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked.

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