ABSTRACT
Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.
Subject(s)
Saccharomyces cerevisiae Proteins , Spliceosomes , Humans , Spliceosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA Splicing , Introns , Ribonucleoprotein, U1 Small Nuclear/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
We have examined the processing of precursor-clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (pre-crRNAs) of the Type I CRISPR-Cas system by incubation of radiolabeled model RNAs with recombinant CRISPR-associated (Cas) endoribonucleases, followed by denaturing polyacrylamide gel electrophoresis (PAGE) of the products. Determination of cleavage position is based on comparison with RNase T1 digestion and base hydrolysis products. The mechanism of cleavage is investigated by chemical and enzymatic characterization of the reaction products as well as by the demonstration that a specific 2'-deoxy substitution 5' to the scissile phosphate blocks endonucleolytic cleavage.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , RNA Cleavage , RNA Precursors/genetics , RNA Precursors/metabolism , CRISPR-Associated Proteins/metabolism , Chemical Precipitation , Chloroform/chemistry , Endoribonucleases/metabolism , Ethanol/chemistry , Hydrolysis , Nucleic Acid Denaturation , Oxidation-Reduction , Phenol/chemistry , RNA Precursors/chemistrySubject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA , Genetic Engineering/methods , RNA, Bacterial , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/physiology , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/physiology , DNA/chemistry , DNA/genetics , DNA/metabolism , Models, Molecular , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolismABSTRACT
Small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in bacteria and archaea are involved in an adaptable and heritable gene-silencing pathway. Resistance to invasive genetic material is conferred by the incorporation of short DNA sequences derived from this material into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by a CRISPR-associated (Cas) RNA endonuclease generates the mature effector RNAs that target foreign nucleic acid for degradation. Here we describe functional studies of a Cas5d ortholog, and high-resolution structural studies of a second Cas5d family member, demonstrating that Cas5d is a sequence-specific RNA endonuclease that cleaves CRISPR repeats and is thus responsible for processing of pre-crRNA. Analysis of the structural homology of Cas5d with the previously characterized Cse3 protein allows us to model the interaction of Cas5d with its RNA substrate and conclude that it is a member of a larger family of CRISPR RNA endonucleases.
Subject(s)
Bacterial Proteins/chemistry , Endoribonucleases/chemistry , Mannheimia/enzymology , RNA Precursors/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Electrophoretic Mobility Shift Assay , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , RNA Cleavage , Repetitive Sequences, Nucleic Acid , Structural Homology, Protein , Substrate SpecificityABSTRACT
In bacteria and archaea, small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by an RNA endonuclease generates the mature effector RNAs that interfere with phage gene expression. Here we describe structural and functional analyses of the Thermus thermophilus CRISPR Cse3 endonuclease. High-resolution X-ray structures of Cse3 bound to repeat RNAs model both the pre- and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene silencing.
