The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding - PubMed (original) (raw)
The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding
Sharon S Kwan et al. RNA. 2005 May.
Abstract
Prp24 is an essential yeast U6 snRNP protein with four RNA recognition motifs (RRMs) that facilitates the association of U4 and U6 snRNPs during spliceosome assembly. Genetic interactions led to the proposal that RRMs 2 and 3 of Prp24 bind U6 RNA, while RRMs 1 and 4 bind U4 RNA. However, the function of each RRM has yet to be established through biochemical means. We compared the binding of recombinant full-length Prp24 and truncated forms lacking RRM 1 or RRM 4 with U6 RNA. Contrary to expectations, we found that the N-terminal segment containing RRM 1 is important for high-affinity binding to U6 RNA and for discrimination between wild-type U6 RNA and U6 with point mutations in the 3' intramolecular stem-loop. In contrast, deletion of RRM 4 and the C terminus did not significantly alter the affinity for U6 RNA, but resulted in the formation of higher order Prp24.U6 complexes. Truncation and internal deletion of U6 RNA mapped three Prp24-binding sites, with the central site providing most of the affinity for Prp24. A newly identified temperature-sensitive lethal point mutation in RRM 1 is exacerbated by mutations in the U6 RNA telestem, as is a mutation in RRM 2, but not one in RRM 3. We propose that RRMs 1 and 2 of yeast Prp24 bind the same central site in U6 RNA that is bound by the two RRMs of human Prp24, and that RRMs 3 and 4 bind lower affinity flanking sites, thereby restricting the stoichiometry of Prp24 binding.
Figures
FIGURE 1.
Recombinant Prp24 proteins. (A) Primary structures of Prp24 N- and C-terminal truncation constructs. The domain structure of the full-length protein, N1234C, is represented schematically at the top with four RNA recognition motifs (RRMs 1–4), the SNFFL box, and the 6xHis tag (H6) at the C terminus. N- and C-terminal residues of the constructs are indicated by position number. The name of each protein truncation construct is indicated at left and the calculated molecular weight (kDa; including the 6xHis tag) is shown at right. (B) Purified Prp24 N- and C-terminal truncation constructs were analyzed by 12% SDS-PAGE and Coomassie Blue staining. The asterisk indicates an unidentified contaminant.
FIGURE 2.
U6 RNA-binding activities of the Prp24 truncation constructs monitored by gel mobility-shift analysis. Complexes and free RNA were resolved on a 6% native polyacrylamide gel. (Lane 1) A control without added protein; the wedges represent increasing protein concentrations, i.e., 50, 100, 200, and 400 nM.
FIGURE 3.
Affinity and stoichiometry of full-length and truncated Prp24 binding to U6 RNA. (A) Binding curves for N1234C (○), N1234 (⋄), N123 (▿), and 234C (▵). The data are from the experiments shown in Figure 2 ▶. Binding of each Prp24 construct was assayed three times, and the deviation from the mean Kd was no more than 10%. (B) Determination of concentration of binding-competent Prp24. A total of 42 nM 32P-labeled U6 RNA was mixed with varying concentrations of N1234C. The fraction of 32P-labeled U6 bound was plotted against the concentration of total N1234C. The linear increase in fraction RNA bound saturates at 1000 nM N1234C. (C) The predicted log molecular weights (in kDa) of protein–RNA complexes in the gel-shift analysis (Fig. 2 ▶) were plotted against the measured Rf values of the complexes. The points were fitted with a straight line (correlation coefficient = 0.98). Symbols are as in A. Numbers located next to the points correspond to the predicted stoichiometry of protein molecules bound to one molecule of U6 RNA.
FIGURE 4.
Binding of full-length and C-terminally truncated Prp24 to truncated and internally deleted U6 RNAs. (A) Proposed secondary structure of S. cerevisiae U6 RNA. Here, the ISL includes residues 62–85, and the telestem includes residues 36–43 and 86–95. Not shown is the potential “central stem” pairing between residues 30–34 and 54–58 (Fortner et al. 1994). The filled arrowheads denote the 5′ ends of the 5′-truncated U6 constructs shown in Figure 4B. Open arrowheads denote the endpoints of the internal deletions of U6 constructs shown in Figure 4C. Nucleotides altered by U6 mutations U6-UA and U6-A79G (see below) are indicated in bold. (B) Gel mobility-shift analysis of 5′-truncated U6 RNAs. The RNA-binding activities of N1234C (lanes 1–4) and N123 (lanes 5–8) for full-length and 5′ end-truncated U6 RNAs as indicated were monitored by gel mobility-shift analysis. Complexes and free RNA (indicated by arrows) were resolved on a 6% native polyacrylamide gel. The wedge represents increasing protein concentrations, i.e., 50, 100, 200, and 400 nM. Complexes are numbered 1 through 3 at the right. (C) Gel mobility-shift analysis of internally deleted U6 RNAs. RNA-binding activities of N1234C (lanes 1–3) and N123 (lanes 4–7) for the internally deleted U6 RNAs U6-Δ32–53 and U6-Δ59–72 were assayed as in B. The wedges represent increasing protein concentrations, i.e., 25, 50, and 100 nM (lanes 1–3,4–6) and 200 nM (lane 7). (D) Gel mobility-shift analysis of U6 RNA oligonucleotides U6-28–87, 45–87, and 45–104. U6 RNA oligonucleotides are depicted by straight lines containing combinations of N123-binding sites I, II, and/or III. RNA-binding activities of N123 for the RNAs were assayed as in B. (Lane 1) A control without added protein; the wedges represent increasing protein concentrations, i.e., 25, 50, 100, 200, 400, and 800 nM (lanes 2–7).
FIGURE 5.
U6-UA and U6-A79G mutations decrease affinity for full-length Prp24, N1234, and N123, but not the RRM 1 deletion construct 234C. (A) Prp24-binding activities of wild-type U6 (lanes 2–6), U6-UA (lanes 7–11), or U6-A79G (lanes 12–16) were monitored by gel mobility-shift analysis. (Lane 1) A control without added protein; the wedges represent increasing N1234C concentrations, i.e., 25, 50, 100, 200, and 400 nM. (B) N1234C-binding curves for wild-type U6 (○), U6-UA (⋄), and U6-A79G (▿). (C) Binding curves for N1234 with wild-type U6 (⋄) and U6-UA (♦), N123 with wild-type U6 (▿) and U6-UA (▾), and 234C with wild-type U6 (▵) and U6-UA (▴). (D) Binding curves for Prp24 constructs as in C, except U6-UA is replaced by U6-A79G. The data are from single representative experiments. Binding of each Prp24 construct was assayed three times, and the deviation from the mean Kd was no more than 10%.
FIGURE 6.
A temperature-sensitive lethal mutation in RRM 1 of Prp24. (A) Sequence alignment of the S. cerevisiae RRMs 1 and 2 with homologous RRMs in H. sapiens and S. pombe. The shaded regions indicate conservation of the physicochemical properties of the residues. The RNP-2 and RNP-1 motifs (Kenan et al. 1991; Burd and Dreyfuss 1994) are underlined. The three residues replaced by alanine substitutions in the RRM1sub allele and a temperature-sensitive mutation in RRM 2 (prp24-R158S) (Vidaver et al. 1999) are indicated. The conserved phenylalanine residue at position 87 in RRM 1 was selected for alanine substitution. (B) Yeast strains containing either the wild-type (WT) or prp24-F87A allele on a low-copy plasmid and a disruption of the genomic PRP24 locus were plated to YEPD medium as serial 10-fold dilutions and incubated at the indicated temperatures and times.
FIGURE 7.
Interaction of mutations in the U6 telestem with mutations in RRMs 1 and 2 of Prp24. Yeast strains with chromosomal disruptions of the Prp24 and U6 RNA genes, and bearing either the wild-type Prp24 allele (PRP24) or temperature-sensitive allele prp24-F87A (RRM 1), prp24-R158S (RRM 2), or prp24-F257I (RRM 3) on a _HIS3_-marked plasmid, as well as wild-type U6 on a counter-selectable _URA3_-marked plasmid, were transformed with plasmids bearing the wild-type U6 allele (U6-wt), U6-U36A,U37A, or U6-A41U,A42U on a _TRP1_-marked plasmid. Starting with cultures of equal cell density, serial 10-fold dilutions were plated to medium containing 5-FOA to select against the wild-type U6 plasmid, and incubated at 25°C for 4 d.
FIGURE 8.
Model for Prp24 interaction with U6 RNA. (Left) The full-length Prp24 molecule is shown binding U6. RRMs 1 and 2 are depicted interacting with site II (nts 47–58), RRM 3 with site I (nts 28–35), RRM 4 with site III (nts 96–103), while the SNFFL box is positioned adjacent to the Lsm-binding site near the 3′ terminus of U6. On the right, three N123 molecules are shown interacting with regions I–III in U6. RRMs 1 and 2 of one molecule bind site II in a similar fashion as for full-length Prp24. It is not clear which RRMs of N123 bind sites I and III.
References
- Brow, D.A. 2002. Allosteric cascade of spliceosome activation. Annu. Rev. Genet. 36: 333–360. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases