Maf1p, a negative effector of RNA polymerase III in Saccharomyces cerevisiae

Abstract

Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1 cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

FIG. 1

Schematic representation of Maf1p and potential orthologs. Abbreviations: Homo sapiens, Hs; Drosophila melanogaster, Dm; Caenorhabditis elegans, Ce; Arabidopsis thaliana, At; Schizosaccharomyces pombe, Sp; Saccharomyces cerevisiae, Sc; Plasmodium falciparum, Pf. Conserved domains are boxed and labeled by letters A (dashed box), B (black box), and C (gray box) in the upper panel. Sequence alignments of each conserved domain (A, B, and C) are shown in the lower panel. Amino acids conserved in at least two sequences are boxed. A star indicates the maf1-1 mutation substituting a stop codon for W319. The two Maf1p-specific signature sequences are underlined.

FIG. 2

Nuclear localization of Maf1p. Δmaf1 (KC3-4D) cells were transformed with the control YCp111 plasmid (A and B) or YCpMAF-4HA, a single-copy plasmid encoding HA-tagged Maf1p (C and D). The cells were stained with DAPI (A and C) or tagged with the 16B12 antibody that recognizes the HA epitope in Maf1p-HA (B and D).

FIG. 3

Mutations in the RPC160 gene are second site suppressors of maf1 mutants. (A) The R16 suppressor (rpc160-16 maf1-1) and its isogenic maf1-1 strain (MB123-2C), The K2 suppressor (rpc160-2 Δmaf), and the isogenic Δmaf strain (KC3-4D) were grown on YPD and replicated on YPD and YPGly prior to incubation at the indicated temperatures. (B) In vivo content of small RNA species in the rpc160-2 mutant carrying the wild-type MAF1 (strain MB156-1B; Table 1) transformed with empty vector ([ ]) or pC160-6 plasmid ([C160]) that complements its cold-sensitive phenotype. Cells were grown in glucose selective medium and shifted to a nonpermissive temperature (2.5 h at 16°C). RNA was extracted and separated by electrophoresis on a 7 M urea–6% PAGE gel using equal amounts of RNA per lane (10 μg). The gel was stained with ethidium bromide.

FIG. 4

tRNA levels are elevated in maf1-1 strains and subsequently reduced in a maf1 rpc160 cold-sensitive suppressor strain. Total RNA was isolated from yeast cells after a shift to a nonpermissive temperature in YPGly (2.5 h at 37°C). (A) Small RNA species from maf1-1 (MT6-7) and the parental strain (T8-1D) were separated on a 7 M urea–6% PAGE gel using equal amounts of RNA per lane (10 μg) and stained with ethidium bromide. (B) Northern analysis of RNA from the wild-type (11D WT), maf1-1 (MT6-7), and parental strain isogenic to maf1-1 (T8-1D) using labeled oligonucleotide probes complementary to U3 RNA, 5S rRNA, tRNA^His, or tRNA^Tyr, as indicated. (C) Northern analysis of RNA from the wild-type (11D WT), maf1-1 (MB123-2C), and maf1-1 strains with a suppressor mutation in C160 (R16) using the probes indicated as in panel B.

FIG. 5

Increased transcription of different Pol III genes in maf1-1 deficient cell extracts. Transcription assays were performed as described in Materials and Methods using a mixture of two genes: a gene transcribed by Pol III and a mini-35S rRNA gene transcribed by Pol I. Transcription was carried out using cell extract (65 μg) from maf1-1 (MT6-7) or parental (T8-1D) strains. RNA was analyzed by urea-PAGE and quantified with a PhosphorImager (Molecular Dynamics). RNA levels transcribed by Pol III were normalized to the level of mini-35S rRNA transcript. (A) Specific transcription of various genes transcribed by Pol III in crude extracts from wild-type (odd lanes) or maf1-1 cells (even lanes). The different DNA templates are indicated. Lane 9, control experiment with no Pol III gene; lane 10, negative control experiment with no added template DNA. (B) Arithmetic means and standard deviations of Pol III transcript levels, corrected for mini-35S rRNA levels, using five independent crude extracts per strain. The different DNA templates are indicated. All of the RNA bands shown in panel A were used for quantification. Gray bars, wild-type crude extract; black bars, maf1-1 crude extracts; AU, arbitrary units.

FIG. 6

In vivo ³²P labeling of RNA. The wild-type (T8-1D) and mutant (MT6-7) cells grown in low-phosphate medium were pulse-labeled for 5 min and supplemented with an excess of nonradioactive phosphate for 15 min to allow for the maturation of 5.8S rRNA. RNAs were extracted and analyzed as described in Materials and Methods. Loading of equal amounts of RNA in each lane was confirmed by staining with ethidium bromide prior to drying of the gel and autoradiography. Synthesis rate of tRNA and 5S rRNA is expressed relative to the 5.8S rRNA species.

FIG. 7

Maf1p interacts with RNA Pol III. Crude extracts from cells expressing HA-tagged C160 (RPC160-HA), myc-tagged Maf1p (MAF1-myc) or both (RPC160-HA MAF1-myc) were incubated with magnetic beads coated with anti-myc (left panels) or anti-HA (right panels) antibodies. The beads were washed, and the bound polypeptides were eluted and analyzed by SDS-PAGE, followed by immunoblotting using specific antibodies, as indicated on the left. For clarity, the polypeptides revealed by the specific antibodies are identified on the right. (A) The beads coated with anti-myc antibodies retained specifically Maf1p-myc; background retention of C160-HA was undetectable. However, C160-HA copurified with Maf1p-myc (left panel). Reciprocally, Maf1p-myc was specifically retained on the anti-HA beads only in the presence of C160-HA (right panel). (B) Pol III subunits C82, AC40, and C34 specifically copurified with Maf1p-myc (left panel), as well as with C160-HA (right panel).