Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21

Abstract

RNA editing within the mitochondria of African trypanosomes is characterized by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The reaction takes place in a high molecular mass ribonucleoprotein (RNP) complex of uncertain composition. Furthermore, factors that interact with the RNP complex during the reaction are by and large unknown. Here we present evidence for an editing-related biochemical activity of the gRNA-binding protein gBP21. Using recombinant gBP21 preparations, we show that the protein stimulates the annealing of gRNAs to cognate pre-mRNAs in vitro. This represents the presumed first step of the editing reaction. Kinetic data establish an enhancement of the second order rate constant for the gRNA- pre-mRNA interaction. gBP21-mediated annealing is not exclusive for RNA editing substrates since complementary RNAs, unrelated to the editing process, can also be hybridized. The gBP21-dependent RNA annealing activity was identified in mitochondrial extracts of trypanosomes and can be inhibited by immunoprecipitation of the polypeptide. The data suggest a factor-like contribution of gBP21 to the RNA editing process by accelerating the rate of gRNA-pre-mRNA anchor formation.

Fig. 1. Recombinant gBP21 stimulates the annealing of gRNAs to cognate pre-edited mRNAs. (A) Schematic representation of gRNA gA6-14Δ16G forming an 11 bp anchor duplex with pre-edited mRNA A6-U5. The gRNA molecule is depicted including a 3′ hairpin loop and a 3′ oligo(U) extension. The length of the RNA strands left and right of the anchor duplex is given in nucleotides. (B) Autoradiogram of a representative annealing experiment with gA6-14Δ16G and radiolabeled A6-U5 pre-mRNA at the r-gBP21 concentrations indicated. Electrophoresis was performed in 8% (w/v) polyacrylamide gels at semi-denaturing conditions (1 M urea). Arrowheads indicate the electrophoretic mobility of the A6-U5 pre-mRNA and the annealed gRNA–pre-mRNA product. Control reactions were set up with 50 nM BSA (instead of r-gBP21) and gND7-506, a non-cognate gRNA (instead of gA6-14Δ16G).

Fig. 2. Verification of the formation of the anchor duplex. (A) Schematic representation of the anchor interaction between gRNA gA6-14Δ16G and pre-mRNA A6-U5. The exact base pairing scheme of the 11 bp anchor is emphasized in addition to the relevant RNase T1 hydrolysis sites on the pre-mRNA (small arrows). The nucleotide length of the expected RNase digestion fragments is given above the gRNA–pre-mRNA hybrid structure. The two 28 and 29 nt long fragments can only form as a result of the annealing reaction. (B) Autoradiogram of a representative experiment. The electrophoretic mobilities of the various RNase T1 fragments shown in (A) are given in the margin on the right. Annotations are as follows: input A6-U5, starting A6-U5 pre-mRNA; –gRNA, –gBP21, control RNase T1 digest minus gRNA and minus gBP21; –gRNA, + gBP21, control digest in the presence of 500 nM gBP21 but without gRNA; +gRNA (100 nM), –gBP21, control digest in the presence of 100 nM gRNA but without gBP21; +gRNA (10 nM), –gBP21, control digest in the presence of 10 nM gRNA and no gBP21; +gBP21, RNase T1 digest in the presence of 10 nM gRNA at varying r-gBP21 concentrations (31, 63, 125, 250 and 500 nM); +BSA, control reaction with 3.8 µM BSA instead of gBP21; +gND7-506, control sample containing gND7-506, a non-cognate gRNA, instead of gA6-14Δ16G.

Fig. 3. Recombinant gBP21 increases the second order annealing rate constant between gRNA and cognate pre-edited mRNA. (A) Determin ation of first order rate constants k₁. Relative signal intensities (SI) of the RNase T1 protected fragments (inserts) were analyzed by densitometry and plotted as a function of the annealing reaction time (t). The data were fitted to the equation SI = 1 – exp(–k₁t). Two representative data sets are shown for gRNA concentrations of 4 and 8 nM. (B) Second order annealing rate constants (k₂) plotted as a function of the gBP21 concentration. k₂ was obtained from k₁ by the equation k₂ = k₁/[gRNA].

Fig. 4. Substrate specificity of the annealing reaction. (A) Annealing reactions were performed with a panel of synthetic complementary RNA reactants (each at 10 nM) capable of forming an identical 15 bp RNA duplex but displaying different overhang topologies. The length of the single-stranded regions is 10 nt in every case and the various annealed structures are depicted above the corresponding autoradiographs. Annealing was performed in the absence and presence of 10 nM r-gBP21. RNA products were separated in 10% (w/v) polyacrylamide gels containing 1 M urea. The electrophoretic mobilities of the annealed RNA products and the input single-stranded reactants (ssRNA) are indicated by arrowheads. (B) Bar graph derived from the densitometric analysis of several experiments as described in (A). The signal intensities of the annealed RNA products were averaged from two to three independent experiments. Error bars represent standard deviations. The level of RNA–RNA ‘self-annealing’ in the absence of r-gBP21 is shown on the right. (C) gBP21-mediated annealing of RNA–DNA and DNA–DNA duplex structures. Results of annealing experiments between identical, base complementary RNA and DNA oligonucleotides of 18 nt length. The reaction was performed at 10 nM r-gBP21 with equimolar concentrations of the RNA and DNA reactants (each at 5 nM). The level of ‘self-annealing’ in the absence of r-gBP21 is given on the right. Error bars represent standard deviations.

Fig. 5. Schematic representation of a panel of gRNA variants to identify the structural requirements of gRNAs required for faithful annealing. All gRNAs are derived from gA6-14Δ16G and are depicted hybridized to A6-U5, the corresponding pre-mRNA. The gRNA variants display the following features: gA6-ΔUTail, the RNA lacks the 3′ oligo(U)-tail; gA6-nostemloop, a 15-nt-long poly(A) sequence replaces the 3′ stem–loop and the oligo(U)-tail; gA6-anchor, just the anchor sequence of gA6-14Δ16G; gA6-ND7stemloop, the original 3′ stem–loop of gA6-14Δ16G is replaced by the stem–loop of gND7-506, a non-cognate gRNA; gA6-anystemloop, the original 3′ stem–loop of gA6-14Δ16G is replaced by an artificial hairpin of five consecutive G–C base pairs.

Fig. 6. Identification of required gRNA substructures for the gBP21-mediated annealing reaction. (A) Annealing reactions were performed without (0) and with r-gBP21 at a concentration of 25 nM. The various samples contain the same radiolabeled A6-U5 pre-edited mRNA (10 nM) in addition to an equimolar concentration of different gRNA variants (see annotation above the different lanes and Figure 5 for structural details). Annealed RNA products (arrowheads) were separated in semi-denaturing (1 M urea), 8% (w/v) polyacrylamide gels. (B) Bar graph derived from the densitometric analysis of two to four independent annealing experiments as shown in (A). The percentage of annealing in the different samples is given relative to the amount of annealed RNA formed between the ‘cognate’ substrates, gA6-14Δ16G gRNA and A6-U5 pre-mRNA, which was set to 100%. Error bars represent standard deviations.

Fig. 7. The 3′ stem–loop element of gND7-506 is necessary and sufficient for the high affinity interaction with recombinant gBP21. (A) Autoradiogram of a representative 3′ boundary experiment with an alkaline hydrolysis ladder of 5′-³²P-labeled gND7-506 RNA (OH^–). Electrophoresis was performed in a denaturing 10% (w/v) polyacryl amide gel. RNA fragments capable of binding to r-gBP21 (+gBP21) contain the entire 3′ stem–loop sequence (annotated as a black bar to the right of the autoradiogram). (B) Representative binding curve between r-gBP21 and the isolated 3′ stem–loop of gND7-506 RNA (see insert). The stability of the stem was enhanced by changing the first base pair of the stem from U/G to C/G. The amounts of bound RNA were determined in a filter binding assay and fitted to the equation [RNA_bound] = n[RNA_free]/(K_d + [RNA_free]). The data represent a K_d of 17 ± 2 nM. Full-length gND7-506 binds to gBP21 with a K_d of 8 ± 2 nM.

Fig. 8. Identification of RNA annealing activities in mitochondrial detergent extracts. Cleared mitochondrial lysates were separated in linear 10–35% (v/v) glycerol gradients and fractionated into 12 fractions of 1 ml. (A) Aliquots from fraction 1 (top of the gradient) to fraction 12 were tested for their annealing activity using gRNA gA6-14Δ16G and radiolabeled A6-U5 pre-mRNA (each at 10 nM) as reactants. Annealing products were separated in semi-denaturing (1 M urea), 8% (w/v) polyacrylamide gels. Arrowheads indicate the electro phoretic mobility of the A6-U5 pre-mRNA and the annealed RNA products. (B) Bar graph derived from the densitometric analysis of the experiment shown in (A). The two annealing activities were named 1st peak and 2nd peak (arrows). Apparent sedimentation coefficients were derived from marker molecules as outlined in Materials and methods. (C) Aliquots from fractions 1–11 were separated in a 12% (w/v) SDS-containing polyacrylamide gel, transferred onto nitrocellulose membranes, and probed for the presence of gBP21 using affinity-purified anti-gBP21 antibodies.

Fig. 9. Immunodepletion and r-gBP21 add-back experiments. The peak fractions of the annealing activities in the 1st peak (fraction 4) and the 2nd peak (fraction 8) were depleted of gBP21 by immunoprecipitation. (A) Western blot analysis to verify the efficiency of the gBP21-depletion reaction. +gBP21, the peak fraction before the immuno depletion; –depl., the gBP21-immunodepleted fraction; Mock, mock-depleted peak fractions. (B) Upper panel: the RNA annealing activity of the gBP21-immunodepleted peak fractions was assayed using the standard annealing assay with gRNA gA6-14Δ16G, radiolabeled A6-U5 pre-mRNA (each at 10 nM) and aliquots (5 µl) of the immunodepleted fractions (1st peak, left panel; 2nd peak, right panel). Annealed RNA products are indicated by arrowheads. Annotations are as in (A). Self-annealing, the level of RNA annealing achieved in the absence of mitochondrial extract. An asterisk represents the annealing product of A6-U5 pre-mRNA with gRNA molecules that have lost their 3′ (U)-tail due to an exonuclease activity within the extract. Lower panel: bar graph derived from the densitometric analysis of the experiment shown in the upper panel. (C) r-gBP21 add-back experiment. A gBP21-depleted fraction of peak 1 was supplemented with r-gBP21 (from left to right: 29, 57 and 143 nM) and tested for its RNA annealing activity as in (B). +gBP21 and –depl. are control samples measuring the RNA annealing activity before and after the immunodepletion, respectively.