scholarly journals Exon and protein positioning in a pre-catalytic group II intron RNP primed for splicing

2020 ◽  
Vol 48 (19) ◽  
pp. 11185-11198
Author(s):  
Nan Liu ◽  
Xiaolong Dong ◽  
Cuixia Hu ◽  
Jianwei Zeng ◽  
Jiawei Wang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Group Ii Intron ◽  
Structural Basis ◽  
Structural Rearrangements ◽  
Spliceosomal Introns ◽  
Structural Impact ◽  
Rnp Complex ◽  
Group Ii ◽  
Structural Insights

Abstract Group II introns are the putative progenitors of nuclear spliceosomal introns and use the same two-step splicing pathway. In the cell, the intron RNA forms a ribonucleoprotein (RNP) complex with the intron-encoded protein (IEP), which is essential for splicing. Although structures of spliced group II intron RNAs and RNP complexes have been characterized, structural insights into the splicing process remain enigmatic due to lack of pre-catalytic structural models. Here, we report two cryo-EM structures of endogenously produced group II intron RNPs trapped in their pre-catalytic state. Comparison of the catalytically activated precursor RNP to its previously reported spliced counterpart allowed identification of key structural rearrangements accompanying splicing, including a remodeled active site and engagement of the exons. Importantly, altered RNA–protein interactions were observed upon splicing among the RNP complexes. Furthermore, analysis of the catalytically inert precursor RNP demonstrated the structural impact of the formation of the active site on RNP architecture. Taken together, our results not only fill a gap in understanding the structural basis of IEP-assisted group II intron splicing, but also provide parallels to evolutionarily related spliceosomal splicing.

scholarly journals Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase

2021 ◽  
Author(s):  
Alfred M. Lentzsch ◽  
Jennifer L. Stamos ◽  
Jun Yao ◽  
Rick Russell ◽  
Alan M. Lambowitz
Keyword(s):  
Active Site ◽  
Rna Binding ◽  
Group Ii Intron ◽  
Structural Features ◽  
Structural Basis ◽  
Template Switching ◽  
Rna Seq ◽  
Cdna Synthesis ◽  
Group Ii ◽  
Template Switch

Reverse transcriptases (RTs) can template switch during cDNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-seq, and may contribute to retroelement fitness by enabling continuous cDNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a template-switching complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template/DNA heteroduplex with a 1-nt 3'-DNA overhang. The latter mimics a completed cDNA after non-templated addition (NTA) of a nucleotide complementary to the 3' nucleotide of the acceptor as required for efficient template switching. The structure showed that the 3' end of the acceptor RNA binds in a pocket formed by an N-terminal extension (NTE) present in non-long-terminal-repeat (LTR)-retroelement RTs and the RT fingertips loop, with the 3' nucleotide of the acceptor base paired to the 1-nt 3'-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine near the RT active site that mediates NTA. Mutation of the latter residue decreased multiple sequential template switches in RNA-seq. Our results provide new insights into the mechanisms of template switching and NTA by RTs, suggest how these reactions could be improved for RNA-seq, and reveal common structural features for template switching by non-LTR-retroelement RTs and viral RNA-dependent RNA polymerases.

Mg2+ vs Ca2+ bound active site of group II intron– A MD study

2020 ◽  
Vol 97 ◽  
pp. 107546
Author(s):  
Abhishek Kumar ◽  
Priyadarshi Satpati
Keyword(s):  
Active Site ◽  
Group Ii Intron ◽  
Group Ii

scholarly journals A glimpse into the active site of a group II intron and maybe the spliceosome, too

RNA ◽  
2008 ◽  
Vol 14 (9) ◽  
pp. 1697-1703 ◽  
Author(s):  
K. T. Dayie ◽  
R. A. Padgett
Keyword(s):  
Active Site ◽  
Group Ii Intron ◽  
Group Ii

scholarly journals A structural analysis of the group II intron active site and implications for the spliceosome

RNA ◽  
2009 ◽  
Vol 16 (1) ◽  
pp. 1-9 ◽  
Author(s):  
K. S. Keating ◽  
N. Toor ◽  
P. S. Perlman ◽  
A. M. Pyle
Keyword(s):  
Structural Analysis ◽  
Active Site ◽  
Group Ii Intron ◽  
Group Ii

scholarly journals A conserved element that stabilizes the group II intron active site

RNA ◽  
2008 ◽  
Vol 14 (6) ◽  
pp. 1048-1056 ◽  
Author(s):  
O. Fedorova ◽  
A. M. Pyle
Keyword(s):  
Active Site ◽  
Group Ii Intron ◽  
Group Ii

scholarly journals Structural insights into the promutagenic bypass of the major cisplatin-induced DNA lesion

2020 ◽  
Vol 477 (5) ◽  
pp. 937-951
Author(s):  
Hala Ouzon-Shubeita ◽  
Caroline K. Vilas ◽  
Seongmin Lee
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Cisplatin Resistance ◽  
Structural Basis ◽  
Abasic Site ◽  
Dna Polymerase Β ◽  
Dna Lesion ◽  
Human Dna ◽  
Optimal Conformation ◽  
Structural Insights

The cisplatin-1,2-d(GpG) (Pt-GG) intrastrand cross-link is the predominant DNA lesion generated by cisplatin. Cisplatin has been shown to predominantly induce G to T mutations and Pt-GG permits significant misincorporation of dATP by human DNA polymerase β (polβ). In agreement, polβ overexpression, which is frequently observed in cancer cells, is linked to cisplatin resistance and a mutator phenotype. However, the structural basis for the misincorporation of dATP opposite Pt-GG is unknown. Here, we report the first structures of a DNA polymerase inaccurately bypassing Pt-GG. We solved two structures of polβ misincorporating dATP opposite the 5′-dG of Pt-GG in the presence of Mg2+ or Mn2+. The Mg2+-bound structure exhibits a sub-optimal conformation for catalysis, while the Mn2+-bound structure is in a catalytically more favorable semi-closed conformation. In both structures, dATP does not form a coplanar base pairing with Pt-GG. In the polβ active site, the syn-dATP opposite Pt-GG appears to be stabilized by protein templating and pi stacking interactions, which resembles the polβ-mediated dATP incorporation opposite an abasic site. Overall, our results suggest that the templating Pt-GG in the polβ active site behaves like an abasic site, promoting the insertion of dATP in a non-instructional manner.

scholarly journals Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Guosheng Qu ◽  
Carol Lyn Piazza ◽  
Dorie Smith ◽  
Marlene Belfort
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Mobile Element ◽  
Housekeeping Genes ◽  
Group Ii Intron ◽  
Conjugative Plasmid ◽  
Group Ii Introns ◽  
Potential Force ◽  
Spliceosomal Introns ◽  
Group Ii

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns.

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