A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Abstract

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.

FIGURE 1.

Structure of I-AniI protein. (A) Linear representation of I-AniI with the two LAGLIDADG motifs highlighted in pink. The location of the conserved basic residues is shown (blue) as is the maturase inactivating substitution (R239E; red). (Top) Full-length I-AniI (F.L.); (middle) the N-terminal fragment (N-Term); (bottom) C-terminal fragment (C-Term). The scale represents 50 amino acids. (B) Three-dimensional structure of I-AniI. The DNA binding surface is composed of curved, green β-sheets shown at the bottom of the structure while the two pink LAGLIDADG α-helices are in the center of the structure. This view emphasizes the possible intron RNA binding surface of the protein. Conserved, surface accessible amino acids are shown as blue spheres; the maturase-deactivating mutation site (R239) is colored red.

FIGURE 2.

Splicing activity of I-AniI, N- and C-terminal proteins. (A) Competition reactions with unlabeled group I introns. 5′-End-labeled A. nidulans COBme pre-RNA (50 nM) was preincubated in buffer with or without competitor RNAs (700 nM) for 10 min and the reaction initiated by the addition of protein (100 nM). The reactions were terminated by the addition of EDTA after 1 min. (Full Length) full length I-AniI; (N-Term) NusA-N-terminal fragment fusion; (C-Term) NusA-C-terminal fragment fusion; (Ns) nonspecific competitor (N. crassa LSU pre-RNA); (S) specific competitor (A. nidulans COBme pre-RNA). (B) Splicing activity of I-AniI and C-terminal protein with a maturase inactivating substitution. 5′-End-labeled A. nidulans COBme pre-RNA (4 nM) was preincubated in buffer and the reaction initiated by the addition of protein (150 nM). The reactions were terminated by the addition of EDTA after 15 min. (F. L.) full-length I-AniI, (F.L.R239E) full-length I-AniI with the 239 arginine to glutamate substitution; (C-Term) NusA-C-terminal fragment; (C-Term R239E) NusA-C terminal fragment fusion with the 239 arginine to glutamate substitution. (Pre) precursor RNA; (LE) ligated exons; (5′E) free 5′ exon. Note that the band above the 5′ exon is generated by cleavage of an alternative 5′SS (U + 2 in the intron). The band above the ligated exons is generated by ligation of this alternative 5′ exon to the 3′ exon.

FIGURE 3.

Splicing kinetics of the C-terminal protein/A. nidulans COBme pre-mRNA complex. A. nidulans COBme pre-RNA (4 nM) was preincubated in buffer at 37°C and the reaction initiated by the addition of protein (500 nM). The plot at the right shows the fraction of A. nidulans COBme pre-RNA remaining versus time. The data were fit to a double exponential with the fast phase describing 90% or 70% of the reactive RNA for the full-length and C-terminal fragment proteins, respectively. The average splicing rate of the C-terminal fragment bound RNA for three independent determinations is 0.61 (±0.14) min⁻¹ while for I-AniI it is 2.1 (±0.48) min⁻¹ (Solem et al. 2002). (Pre) precursor RNA; (LE) ligated exons; (5′E) free 5′ exon.

FIGURE 4.

Binding of I-AniI, N-, and C-terminal fragment proteins. (A) Equilibrium binding. Trace amounts of ³²P-labeled A. nidulans COBme pre-RNA were incubated with a range of protein concentrations and filtered. The data were fit to: Fraction RNA bound = A × E/(E + K_d^app) where A is the total amplitude bound and E is the concentration of I-AniI and K_d^app is the apparent equilibrium dissociation constant. The experiment was repeated three times to yield a K_d^app of 12.3 (±2.1) nM and 50 (±17) nM for the full-length I-AniI and C-terminal protein, respectively. Note that the filter retention efficiency of the C-terminal fragment in this assay (~10%) causes the amplitude to be approximately threefold less than that for the full-length protein. (B) k_off determination for the C-terminal protein. Dissociation of the A. nidulans COBme pre-RNA/protein complexes was initiated by dilution in RNA binding buffer containing an 800-fold molar excess of unlabeled to labeled A. nidulans COBme pre-RNAs followed by filtration at the times indicated. The data for the C-terminal protein were fit to a double exponential with 68% of the complex dissociating rapidly and 32% dissociating more slowly. The dissociation rate constant was determined from two independent experiments. The k_off of the fast phase is 0.20 (range 0.18–0.21) min⁻¹ and for the slow phase is 0.01 (range 0.009–0.01) min⁻¹. A plot of an experiment with the full-length I-AniI performed at the same time is shown as a control. (C) Turnover kinetics of the splicing reaction. A. nidulans COBme pre-RNA (270 nM) was incubated in buffer and the reaction initiated by the addition of protein (90 nM). Under these conditions, the protein is saturated with pre-RNA substrate and the observed rate of splicing reflects primarily the dissociation of the complex (Ho and Waring 1999; Solem et al. 2002). The average splicing rate of the C-terminal fragment bound RNA for three independent determinations is 0.004 (±0.002) min⁻¹ while for I-AniI it is 0.01 (range 0.02–0.008) min⁻¹ (Solem et al. 2002).

FIGURE 5.

Ribonuclease-mapping of I-AniI and C-terminal protein RNPs. (A) Representative footprinting experiment. 5′-End-labeled A. nidulans COBme pre-RNA (13 nM) was cleaved with RNase 1 or V1 in the absence or presence of the full-length (F.L.) or C-terminal fragment (C-Term) protein. Sites of cleavage sites were deduced by comparison of the products against RNA ladders generated by partial alkaline hydrolysis (OH) or partial digestion with ribonuclease T1. To control for degradation, one set of samples was treated without added ribonuclease (minus (−) RNase). (B) Summary of ribonuclease protection/enhancement in the presence of either full-length or C-terminal protein. The secondary structure model of A. nidulans COBme pre-RNA shows protection from ribonuclease 1 (green shading) or V1 (red shading) digestion while a red arrow indicates enhanced V1 cleavage in the presence of either protein. Blue (V1) or green (1) arrows show cleavages unchanged by protein binding. Only consistent cleavages/protections observed from two independent experiments are shown.