CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains.
Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.
Ostersetzer
Oren
O
Department of Biology and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Cooke
Amy M
AM
Watkins
Kenneth P
KP
Barkan
Alice
A
D001665:Binding Sites / Q000235:genetics; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry; D007438:Introns / Q000235:genetics*; D008274:Magnesium / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D000072260:RNA Splicing Factors; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism
C436683:CRS1 protein, Zea mays; D009687:Nuclear Proteins; D010940:Plant Proteins; D000072260:RNA Splicing Factors; D016337:RNA, Catalytic; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D017665:Hydroxyl Radical; D008274:Magnesium