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. 1999 Oct;19(10):6543-53.
doi: 10.1128/MCB.19.10.6543.

Genetic and physical interactions involving the yeast nuclear cap-binding complex

P Fortes  1 , J Kufel, M Fornerod, M Polycarpou-Schwarz, D Lafontaine, D Tollervey, I W Mattaj
Affiliations

Genetic and physical interactions involving the yeast nuclear cap-binding complex

P Fortes et al. Mol Cell Biol. 1999 Oct.

Abstract

Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.

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Figures

FIG. 1
FIG. 1
yCBC affects vegetative growth rate. Strain YJV159 (wild type) was disrupted for GCR3 (cbp80-Δ), for MUD13 (cbp20-Δ), or for both (cbp20/80-Δ). These strains were grown to mid-log phase, and four dilutions of each were plated to compare the growth rates. The doubling time of each strain was also assayed in liquid culture and is indicated on the right.
FIG. 2
FIG. 2
yCBC interacts with Mud2p and with Mud10p. [35S]methionine-labeled Mud2p/Luc2p (lanes 1 to 5), Snu71p/Luc5p (lanes 6 to 10), and Mud10p/Luc4p (lanes 11 to 15) were incubated with a control column (MOCK) or with a yCBC column as indicated. Samples were fractionated into nonbound supernatant (S) and bound pellet (P) fractions and analyzed by SDS-polyacrylamide gel electrophoresis. In lanes 1, 6, and 11, 25% of the input protein was loaded.
FIG. 3
FIG. 3
Growth of the LUC8 and LUC9 strains carrying functional CBC. Dilutions (1- to 102-fold) of luc8 and luc9 strains along with the wild-type isogenic (WT) control strain were spotted on minimal plates at 16, 23, 30, and 37°C and incubated for 3 days.
FIG. 4
FIG. 4
The yeast pre-rRNA processing pathway. (A) Structure of the pre-rRNA with positions of oligonucleotides used for hybridization. In the 35S pre-rRNA, the mature 18S, 5.8S, and 25S rRNA sequences are flanked by the 5′ and 3′ external transcribed spacers (5′ ETS and 3′ ETS) and separated by internal transcribed spacers 1 and 2 (ITS1 and ITS2). (B) Major pre-rRNA processing pathway in yeast. Note that a minor alternative pathway in ITS1 generates an alternative form of 5.8S rRNA (5.8SL) that is extended 5′ to site BIL (not shown). (C) Structures of the aberrant 23S and 21S RNAs.
FIG. 5
FIG. 5
Northern analysis of pre-rRNA (A and C) and snoRNA (B and D) levels in LUC8 and LUC9 strains. RNA was extracted following growth at 23°C and 18 h after transfer to 37°C (A and B) or following growth at 30°C and 12 h after transfer to 16°C (C and D). The oligonucleotide probes used in panels A and C were 003 (top) and 002 (bottom). WT, wild type.
FIG. 6
FIG. 6
yCBC is required for normal pre-rRNA processing. For Northern blot analysis of mature and precursor rRNAs, RNA was extracted from wild-type (WT) and cbp strains as indicated. (A) Hybridization with a probe complementary to the mature 18S and 25S RNAs; (B) hybridization with a probe complementary to the 5′ region of ITS1 (oligonucleotide 002); (C) hybridization with a probe complementary to the 3′ region of ITS1, downstream of site A3 (oligonucleotide 001); (D) hybridization with a probe complementary to the central region of ITS1, between sites A2 and A3 (oligonucleotide 003); (E and F) hybridization with a probe complementary to the 5′ region of ITS2 (oligonucleotide 013). (G) Ratios of steady-state levels of mature 18S and 25S rRNAs. The positions of mature and precursor rRNA species are indicated; 21S and 20S pre-rRNAs are not well resolved; the identity of the 32S intermediate was verified by hybridizing a riboprobe complementary to the region between sites A0 and A1. Positions of the oligonucleotide probes are depicted in Fig. 4.
FIG. 7
FIG. 7
yCBC does not affect accumulation of various snoRNAs. For Northern blot analysis of low-molecular-weight RNAs, RNA was extracted from wild-type and cbp strains as indicated. The probes used for hybridization are described in Materials and Methods.
FIG. 8
FIG. 8
yCBC affects steady-state levels of mRNAs of ribosomal proteins. RNA was extracted from either wild-type or cbp strains as indicated. Additionally, control RNA was extracted from a temperature-sensitive-lethal splicing-deficient prp2-1 strain grown either at the permissive temperature (25°C) or after shift to the nonpermissive temperature (37°C) for 60 min. (A) Analysis of RPS and RPL mRNAs and pre-mRNAs. The positions of mature and precursor mRNAs are indicated. Probes used for hybridization are described in Materials and Methods. (B) Sequences at the 5′ splice sites of pre-mRNAs analyzed in this study. Nonconsensus residues are underlined. All pre-mRNAs tested contain one intron, and in all cases it is located close to the 5′ end of the pre-mRNA.
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