Supplementary Online Material

Nucleolar clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complex

Haeusler RA, Pratt-Hyatt M, Good PD, Gipson T, and Engelke DR

Methods

Microtubule component genes tested for effects on tgm silencing

Tested gene deletions include Ddyn2, Djnm1, Dkip1, Dkip2, Dnip100, Dpac11, along with several other gene deletions and mutations tested previously, such as Dcik1, Dcin8, Dtub3, arp7 and arp9 mutants, and several more (Wang et al. 2005). We also titrated cell growth in the presence of the microtubule depolymerizing drug, nocodazole, to identify any non-lethal concentrations that released tgm silencing, but all concentrations that allowed growth also allowed silencing.

RNA isolation and northern blot

RNA isolation using hot acid phenol was adapted from (Kohrer and Domdey 1991). RNAs were separated on denaturing 6% or 12% polyacrylamide gel, transferred to Nytran membrane, and probed with radiolabeled DNA oligonucleotides complimentary to the 10 tRNALeu(CAA) genes (5’-TGCTAAGAGATTCGAACTCTTGCA-3’) or 100-200 5S rRNA genes (5’- TTCGCGTATGGTCACCCACTACA-3’). Detection was performed with a PhosphorImager (Molecular Dynamics 445 SI) or Typhoon, and quantified with IPlab Gel (Signal Analytics) or ImageQuant software.

Primer extension analysis
DNA oligonucleotides (Invitrogen) complementary to the HIS3 reporter mRNA (5’-GCTCATTTTTGGAATTG-3’) and ADH1 mRNA (5’-CGTAGAAGATAACACCT-3’) were 5’ end labeled gamma-32P-ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs). Primer extensions were carried out as previously described (Lee et al. 1991). Radioactive signals were visualized using the Typhoon Trio+ and quantitated using ImageQuant 5.2 (GE Healthcare).

Figure S1. In situ hybridization of unsynchronized cells. Undivided nuclei are shown from unbudded (A) and budded (B) cells, where state of budding was determined from DIC images of the cells. Dividing nuclei are shown in (C). Fluorescent oligonucleotide probes complementary to the U14 snoRNA (green) or 10 tRNALeu(CAA) genes (red) were used for hybridization. Blue represents DAPI staining of nucleoplasmic DNA. tRNA gene signal (red) consistently overlapped the nucleolar signal (green) prior to and throughout division of the nucleus. S. cerevisiae undergoes a closed mitosis, in which both the nucleus and the nucleolus remain intact (Loidl 2003), and staining of nucleolar proteins and ribosomal DNA (rDNA) throughout mitosis has indicated that this region is one of the last in the genome to divide (Fuchs and Loidl 2004; Machin et al. 2005).


Figure S2. RNA analysis. (A) Northern blot analysis of total RNA isolated from yeast left untreated (n=7), treated for 1 hour (n=8), or arrested in nocodazole (n=9). A probe to the intron of tRNALeu(CAA) reveals that pre-tRNAs are still synthesized after microtubule depolymerization with nocodazole. The ratio of the precursors (precursor tRNA + partially processed intermediate) to mature tRNA decreases slightly after prolonged exposure to nocodazole, indicating a slight reduction in transcription. (B) Primer extension of a HIS3 reporter gene on a tgm silencing test construct with an active (+) or inactive (-) tRNA gene. The ratio of HIS3 to ADH1 signals from both constructs decrease after extended nocodazole treatment, indicating that the tgm silencing initiated by the tRNA gene is not released.

Figure S3. Deletion of TOP2 does not release tgm silencing. Strains in which two of the topoisomerases, top1∆ and top3∆) had been deleted had already been tested to determine whether they released tgm silencing (Wang et al., 2005b). Since TOP2 is known to affect nucleolar function and architecture, it was tested here as a negative control. Strain containing mutation in a top2-4 mutation (gift of Frank Uhlmann) (Holm et al., 1985) is tested for silencing of a HIS3 reporter adjacent to an active tRNA gene as in Figure 2. No His+ growth was observed at either 25°C or 30°C (shown; at higher temperatures growth is compromised on His+ medium). The positive control strain contains mod5∆ deletion that releases silencing (Pratt-Hyatt et al, submitted; see also Figure 2 for further positive controls).

Figure S4. . Effects of condensin mutations to the transcription and processing of tRNALeu3. Northern analysis was performed to analyze the ratio of mature:precursor tRNAs as a measure of serious effects on transcription or nucleolytic processing (5’ and 3’ ends, intron removal; Upadhya et al., 2002) in the condensin ts mutants Northern blots of total cellular RNA (5µg/lane) were probed with a 32P-labeled oligodeoxynucleotide complementary to the mature domain of tRNALeu3 (Lee et al., 1997). RNA was extracted from cells grown at 30º in triplicate to an OD of 0.6-0.7 then incubated for the indicated times (15 or 30 minutes) at 30º or 37oC. Triplicate signals from blots were quantified by Typhoon for both the mature and precursor forms of the tRNA.

. Higher mature:precursor ratios indicate decreased synthesis of new transcripts by pol III relative to processing, while lower ratios would indicate decreased processing rates relative to transcription. The condensin mutants gave modestly higher ratios consistent with mildly decreased transcription or increased processing, but the changes were not of sufficient magnitude to allow confident interpretation. The growth of these previously described mutant strains was slowed even at permissive temperatures, which in itself can alter the mature:precursor ratios by this magnitude (unpublished results).

Figure S5. Nocodazole treatment causes microtubule depolymerization. In untreated cells, spindle microtubules are clearly visible (green). After treatment with 15 µg/ml nocodazole for one hour or several hours, no microtubule structures can be seen, although a single spot of tubulin sometimes remains after a treatment of only one hour, similar to published observations (Jacobs et al., 1988). Nuclei are also stained with DAPI (blue).

Figure S6. DMSO treatment alone does not disrupt nucleolar structure. Dimethyl sulfoxide (DMSO) (at a final concentration of 0.21%, higher than the 0.15% in Figure 3 experiments) was added directly to yeast media during active growth, 1 hour prior to fixation. Yeast were subsequently used for in situ hybridization, probing for tRNALeu3 genes (upper panels) and pre-tRNALeu3 (lower panels). Representative cells are shown Deconvolution microscopy revealed the nucleolar localization of both tRNA genes and pre-tRNAs, indistinguishable from wild type (Figure 3), consistent with previous work on DMSO effects on microtubule structures (Jacobs et al. 1988).


Fuchs, J. and Loidl, J. 2004. Behaviour of nucleolus organizing regions (NORs) and nucleoli during mitotic and meiotic divisions in budding yeast. Chromosome Res 12(5): 427-438.

Holm, C., Goto, T., Wang, J.C., and Botstein, D. (1985) DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41, 553-563.

Jacobs, C.W., Adams, A.E., Szaniszlo, P.J., and Pringle, J.R. 1988. Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J Cell Biol 107: 1409-1426.

Kohrer, K. and Domdey, H. 1991. Preparation of high molecular weight RNA. Methods Enzymol 194: 398-405.

Lee, J.Y., Rohlman, C.E., Molony, L.A., and Engelke, D.R. 1991. Characterization of RPR1, an essential gene encoding the RNA component of Saccharomyces cerevisiae nuclear RNase P. Mol Cell Biol 11: 721-730.

Lee, Y., Kindelberger, D.W., Lee, J.Y., McClennen, S., Chamberlain, J., and Engelke, D.R. (1997) Nuclear pre-tRNA terminal structure and RNase P recognition.. RNA 3: 175-185.

Loidl, J. 2003. Chromosomes of the budding yeast Saccharomyces cerevisiae. Int Rev Cytol 222: 141-196.

Machin, F., Torres-Rosell, J., Jarmuz, A., and Aragon, L. 2005. Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase. J Cell Biol 168: 209-219.

Upadhya, R., Lee, J., and Willis, I.M. (2002). Maf1 is an Essential Mediator of Diverse Signals that Repress RNA Polymerase III Transcription. Mol. Cell 10: 1489-1494.

Wang, L., Haeusler, R.A., Good, P.D., Thompson, M., Nagar, S., and Engelke, D.R. 2005. Silencing near tRNA genes requires nucleolar localization. J Biol Chem 280: 8637-8639.