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Druggability
splice
The splicing of a bacterial group II subclass B intron B.me.I1 from Bacillus megaterium chromosomes was investigated. RT-PCR and nucleic acid hybridization methods were used to understand the role of the intron-encoded protein (IEP) in the splicing of B.me.I1. An in vivo assay showed that the splicing occurred in the absence of IEP. An in vitro assay showed that B.me.I1 was spliced under conditions similar to those of the intracellular environment with no help from other biological molecules. Because all group II introns previously reported needed IEPs for their splicing in vivo, our results suggest that B.me.I1 is an "actual" self-splicing group II intron. This is also the first report to recognize the existence of group II introns that independently splice mRNA in vivo. The self-splicing of a bacterial intron may support that eukaryotic spliceosomal introns originated in bacterial genomes.
Group II intron
Minor spliceosome
Bacillus megaterium
Splicing factor
Bacterial genome size
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The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.
Group II intron
Splice site mutation
Splicing factor
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We have studied the effect of the 5' cap structure on the splicing of precursor mRNAs containing three exons and two introns within a single molecule in a HeLa nuclear extract. When a precursor mRNA was capped, the upstream intron was spliced out more efficiently than the downstream intron. The differential splicing reactions of the two introns are not due to differences in the intrinsic efficiency of splicing of each intron, since the preferential excision of the upstream intron was also observed when the positions of the two introns relative to the cap structure were reversed. When uncapped precursor mRNA was used as substrate, the downstream intron was spliced out appreciably, but splicing of the upstream intron was greatly reduced. Preincubation of the extract with cap analogues inhibited splicing of the upstream intron but not the downstream intron. Thus, the cap structure exerts its effect primarily on the 5' proximal intron.
Group II intron
Precursor mRNA
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Group II intron
Minor spliceosome
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Intron 1 of the coxl gene of yeast mitochondrial DNA (al1) is a group HA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, al1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4CI or KCI or 2M (NH4)2SO4) for ready detection of splicing activity. In KCI-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4CI or KCI, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AM RNA is most reactive at 37–42°C, as compared with 45°C for the group IIB Introns; and it lacks the KCl-or NH4CI-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron al5γ, the domain 4 of ah can be largely deleted in cis, without blocking splicing; also, frans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.
Group II intron
Protein splicing
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Group II intron
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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.
Group II intron
Exonic splicing enhancer
SR protein
Splicing factor
Polypyrimidine tract
Spliceosome
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Group II intron
Splicing factor
Exonic splicing enhancer
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The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.
Group II intron
Splice site mutation
Splicing factor
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Citations (50)