An RNA Binding Motif in the Cbp2 Protein Required for Protein-stimulated RNA Catalysis

Abstract

The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing. Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron. In vitro, this mitochondrial intron can be made to selfsplice or undergo protein-facilitated splicing by varying the Mg 2؉ and monovalent salt concentrations. This twocomponent system, therefore, provides a good model for understanding the role of proteins in RNA folding. A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions. A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing. Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing. Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant. This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing.The terminal intron (intron 5) of the yeast gene encoding cytochrome b (COB) can self-splice in vitro at high concentrations of Mg 2ϩ (1, 2), but the protein encoded by the nuclear CBP2 gene is essential for splicing at physiological concentrations of Mg 2ϩ (3). Although this group IA intron possesses the conserved secondary and tertiary structures found in all group I introns, it varies in important ways from the prototype Tetrahymena rRNA intron (4). In particular, the internal guide sequence that establishes the substrate specificity starts 220 nucleotides downstream of the 5Ј splice junction, rather than the usual 14 -20 nucleotides found in most other group I introns. The intron is AU-rich, and requires higher levels of Mg 2ϩ for stabilization of the active structure than the GC-rich Tetrahymena group I intron. Like other group IA introns, the fifth intron of COB pre-mRNA (bI5) is also devoid of the peripheral RNA element, P5abc, that is important for stabilizing the catalytic core of the Tetrahymena rRNA intron (5). It is therefore conceivable that Cbp2 compensates for this RNA structure and stabilizes its RNA partner by contributing substantial binding energy in a manner similar to the CYT-18 protein of Neurospora (6, 7). UV cross-linking, chemical, and enzymatic modification studies indicate that Cbp2 contacts bI5 at multiple sites in the catalytic core and peripheral RNA elements and stimulates the formation of the catalytically active structure (8, 9). Based on these and kinetic studies, Weeks and Cech (10) proposed that Cbp2 serves as a ter...

Recruitment of a peptidyl-tRNA hydrolase as a facilitator of group II intron splicing in chloroplasts

“…The gray bar highlights a putative RNA‐binding region that is found in predicted CRS2 proteins in plants, but not in their PTH ancestor. The lower box shows an alignment of this region of predicted plant CRS2s with a region of the yeast protein Cbp2 that has been implicated in binding group I introns and in facilitating group I intron splicing (Tirupati et al ., 1999). The C‐terminal sequences of the presumed CRS2 orthologs in iceplant (accession No.…”

“…Among the four plant species for which CRS2 C‐terminal sequence is available, there is some variation within this extension, but all variants maintain the pattern of basic and aromatic residues (box, Figure 4). This extension resembles a six‐amino‐acid motif (RYRYKF) in the group I intron splicing factor Cbp2, which contacts intron RNA and is essential for Cbp2 to promote splicing (Tirupati et al ., 1999) (box, Figure 4). We hypothesize that the acquisition of this C‐terminal extension was an essential step in the evolution of CRS2 from its PTH ancestor.…”

Two Distinct Binding Modes of a Protein Cofactor with its Target RNA

“…13,17 This RNA structural-destabilizing activity likely arises from the electrostatic interactions between the negatively charged RNA and the positively charged domains of CBP2, 42 which tend to destabilize RNA-RNA contacts to optimize RNA-protein contacts. It has been postulated that specific protein cofactors might first use non-specific electrostatic interactions to destabilize misfolded RNA structures, chaperoning the RNA to its native state, which is then Figure 10.…”

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen

“…The af®nity of HDAg peptides to various nucleic acids was examined by the double-®lter binding assay (32,33). The RNP complexes were generated by incubating a ®xed concentration of 32 P-labeled nucleic acid with increasing concentrations of HDAg peptide (0±10 mM) for 30 min at 37°C in the standard binding buffer [40 mM Tris±HCl (pH 7.5), 0.1 M NaCl, 0.02 mM EDTA, 2% glycerol and 40 mg/ml bovine serum albumin] in a total volume of 50 ml.…”