Supplemental Material The poly(A) binding protein Nab2 functions in RNA polymerase III transcription L. Maximilian Reuter, Dominik M. Meinel, Katja Sträßer Supplemental Figure S1. Nab2 occupies all RNAPIII transcribed genes. (A-F) Individual traces of Rpb3 (RNAPII), Rpc160
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Supplemental Material
The poly(A) binding protein Nab2 functions in RNA polymerase III transcription
L. Maximilian Reuter, Dominik M. Meinel, Katja Sträßer
TFC1-TAP MATa; TFC1-TAP::TRP1; ade2-1; his3-11,15; ura3-52; leu2-3,112; trp1-1; can1-100; GAL++ this study
TFC8-HA MATa; TFC8-HA::HIS3mx6; ade2-1; his3-11,15; ura3-52; leu2-3,112; trp1-1; can1-100; GAL+ this study
Supplemental Table S2. Plasmids used in this study.
Plasmid Description Source
pAC1038 ∆N-NAB2-GFP, CEN, LEU2 (Green et al. 2002)
pAC1152 ∆N-NAB2 (nab2-1), CEN, LEU2 (Marfatia et al. 2003)
pAC2307 nab2-C437S, CEN, LEU2 (Kelly et al. 2007)
pGex-6P-1-NAB2 the coding region of NAB2 was amplified by PCR creating BamHI and NotI sites and cloned into the same sites of pGex-6P-1
this study
pBluescriptIIKS-SNR6 the genomic SNR6 locus including 117 upstream and 253 downstream was amplified by PCR creating EcoRI and XbaI sites and cloned into the same site of pBluescriptIIKS
(Brow and Guthrie 1990)
pBluescriptIIKS-tA the coding region of tRNAAla(UGC)E was amplified this study
by PCR creating NotI and XbaI sites and cloned into the same sites of pBluescriptII KS
pBS1479 plasmid for genomic TAP-tagging with the TRP1-KL marker
Euroscarf
pET21a-BDP1 the coding region of BDP1 was amplified by PCR creating EcoRI and NotI sites and cloned into the same sites of pET21a
this study
pET21a-BRF1 the coding region of BRF1 was amplified by PCR creating SacI and NotI sites and cloned into the same sites of pET21a
this study
pET21a-NAB2 the coding region of NAB2 was amplified by PCR creating SacI and NotI sites and cloned into the same sites of pET21a
this study
pET21a-TBP the coding region of TBP was amplified by PCR creating EcoRI and NotI sites and cloned into the same sites of pET21a
this study
pRS315-NAB2 the ORF of NAB2 including 532 bp 5’ and 311 bp 3’ was amplified by PCR creating Not1 and Xho1 sites and cloned into the same sites of pRS315
this study
pRS315-nab2-34 nab2-34 inserted by homologous recombination after mutagenic PCR of the NAB2 ORF
this study
pRS316-NAB2 the ORF of NAB2 including 532 bp 5’ and 311 bp 3’ was amplified by PCR creating Not1 and Xho1 sites and cloned into the same sites of pRS316
this study
pYM15 plasmid for genomic HA tagging with the HIS3mx6 marker
Euroscarf
Supplemental Table S3. dsDNA probe sequences used in this study.
For quantification of the purified DNA, the Step One Plus cycler (Applied Biosystems) was
used with the Applied Biosystems Power Sybr Green PCR Master Mix. For calculation of the
enrichments, a non-transcribed region (NTR) on Chromosome V (174131-174200) was
applied. To determine the primer efficiencies, standard curves were used. Occupancies were
calculated as enrichments of the tested gene over the NTR with the following formula:
(E^(CTIP-CT
INP))NTR/(E^(CTIP-CT
INP)YFG). Regions amplified by the respective primer pairs are:
SNR6 (chromosome XII, 366249-366328), tDNALys (amplification of 21 genes encoding
tRNALys(CUU), .e.g. chromosome III, 151295-151354), RPR1 (chromosome V, 117975-
118091) and SCR1 (chromosome V, 441987-442508, primers as shown in Supplemental
Figure S5A and described in (Ghavi-Helm et al. 2008)).
Oligo design for EMSAs
Used oligos were annealed in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, heated
to 95°C for 5 min and slowly cooled to room temperature (45 min). dsDNA code: (+): 4-
nucleotide mismatches; (-): no mismatches; 1: no double nucleotides; 2: double nucleotides
allowed. Combinations of oligo sequences were as follows:
dsDNA1(+): dsDNA1(+)_1 and dsDNA1(+)_2;
dsDNA1(-): dsDNA1(+)_1 and dsDNA1(-)_2;
dsDNA2(+): dsDNA2(+)_1 and dsDNA2(+)_2;
dsDNA2(-): dsDNA2(+)_1 and dsDNA2(-)_2
Supplemental References
Brow DA, Guthrie C. 1990. Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes & Development 4: 1345-1356.
Ghavi-Helm Y, Michaut M, Acker J, Aude JC, Thuriaux P, Werner M, Soutourina J. 2008. Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription. Genes & development 22: 1934-1947.
Green DM, Marfatia KA, Crafton EB, Zhang X, Cheng X, Corbett AH. 2002. Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p. The Journal of biological chemistry 277: 7752-7760.
Kelly SM, Pabit SA, Kitchen CM, Guo P, Marfatia KA, Murphy TJ, Corbett AH, Berland KM. 2007. Recognition of polyadenosine RNA by zinc finger proteins. Proceedings of the National Academy of Sciences of the United States of America 104: 12306-12311.
Marfatia KA, Crafton EB, Green DM, Corbett AH. 2003. Domain analysis of the Saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, Nab2p. Dissecting the requirements for Nab2p-facilitated poly(A) RNA export. The Journal of biological chemistry 278: 6731-6740.
Meinel DM, Burkert-Kautzsch C, Kieser A, O'Duibhir E, Siebert M, Mayer A, Cramer P, Soding J, Holstege FC, Strasser K. 2013. Recruitment of TREX to the transcription machinery by its direct binding to the phospho-CTD of RNA polymerase II. PLoS Genet 9: e1003914.
Zaros C, Thuriaux P. 2005. Rpc25, a conserved RNA polymerase III subunit, is critical for transcription initiation. Molecular microbiology 55: 104-114.