Supplementary. Table 1: Oligonucleotides and Plasmids. complementary to positions from 77 of the SRα '- GCT CTA GAG AAC TTG AAG TAC AGA CTG C

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1 Supplementary Table 1: Oligonucleotides and Plasmids '- GCT CTA GAG AAC TTG AAG TAC AGA CTG C '- CCC AAG CTT ACA GTG TGG CCA TTC TGC TG '- CGA CGC GTA CAG TGT GGC CAT TCT GCT G complementary to positions from 719 of the SRα coding region, including a XbaI site (underlined); used for PCR amplification of the 498 bp fragment for cloning into the pjm326 and plew100 vectors, respectively, to generate the SRα-hp-1 construct. complementary to positions from 1198 of the SRα coding region, including a HindIII site; used for PCR amplification of the 498 bp fragment for cloning into the pjm326 vector to generate the SRα-hp-1 construct. complementary to positions from 1198 of the SRα coding region, including a MluI; used for PCR amplification of 498 bp fragment for cloning into the plew100 vector to generate the SRα-hp-1 construct. Sra-newhind 5 - CCC AAG CTT TGC ACT TCA CCT TCA CCT TGC complementary to positions from 490 of the SRα coding region, including a HindIII site; used for PCR amplification of the 414 bp fragment for cloning into the pjm326 vector to generate the SRα-hp-2 construct Sra-newmluI 5 - CCC ACG CGT TGC ACT CAC CTT CAC CTT GC complementary to positions from 490 of the SRα coding region, including a MluI; used for PCR amplification of 414 bp fragment for cloning into the plew100 vector to generate the SRα-hp-2 construct Sra-newxbaI 5 - CCC TCT AGA GCT CAA TAA TTA ATC AGG TC complementary to positions from 77 of the SRα coding region, including a XbaI site (underlined); used for PCR amplification of the 414 bp fragment for cloning into the pjm326 and plew100 vectors, respectively, to generate the SRα-hp-2 construct AD ACA AAG CTT AGG AGT GAC GTT GGC ATT TC complementary to from position 478 of the tsnap42 coding region, including a hind III site; used for PCR amplification of the 497 bp fragment for cloning into the pzjm vector, to generate the tsnap42 construct. AD ACA CTC GAG CCA ATT GAA GGT GGT ACG CT complementary to from position 804 of the tsnap42 coding region, including a xho I site; used for PCR amplification of the 497 bp fragment for cloning into the pzjm vector, to generate the tsnap42 construct '- CCG CTC GAG AGC CGG AGC GCA TTG CTC TG complementary to positions 1-16 of 7SL RNA '- CCC AAG CTT CCG CCT CGC GAC GAC ACT TG complementary to positions of 7SL RNA

2 '- TTC GAG AGA TAT AGC T complementary to positions of U6 TB-U3 5'- GTT CGA TGA ACG GCA complementary to positions of U '- TCT CCT CAC CCC CTC GCG complementary to positions 3-20 of tubulin '- ATG CAG ATA GCC TCA CGC complementary to positions of tubulin '- GTA GCG ATG CTG ACG TGC AA complementary to positions of 28S rrna '- CAC TCA CAC ACA CAT GGC TAT complementary to positions of 28S rrna '- AAC TAA CGC TAT TAT TA complementary to positions 1-17 of SL RNA '- GCA GGA ACC AAC AGC ACA ATG CG complementary to positions of SL RNA Sm03 5'- CCA TCC CCG TGC ATG CCA CAT TTC TCA GTG TCG complementary to positions of 5.8S rrna 1222B12 5'- CCG CTC GAG TAG GGA GCA TCC ACA TGA A complementary to positions of fibrillarin 1222D12 5'CCC AAG CTT GTT GTA CTG CTT TCT TCA TA complementary to positions of fibrillarin '- GAT CAC TGC CGG TGG TAT CT complementary to positions of vhppase '- GCA CCG AAA TGT AAC CGA GT complementary to positions of vhppase '- ATT TTT CCA TCG GCT TTG TTG '- ACC CAA AAC TCA TTC GTT GC '- TCC CTT TAT CTG CTC GCT GT complementary to positions of hexose transporter complementary to positions of hexose transporter complementary to positions of EP '- TAG AAT GCG GCA ACG AGA C complementary to positions of EP

3 1106 5'- CTT AGC CAT GCA TGC CTC complementary to positions of 18S rrna LC-M4-1S 5 - GTC CCT CTC CAA ACG GAG A complementary to positions 1-19 of srrna4 LC-M4-115AS 5 -GTC CCT GAG CAT GAA ATT complementary to positions of srrna4 Sh3-sl-neg 5 -CCA TAA CGG CTT AAG CAC AAG complementary to positions upstream of SL RNA Sh4-sl-neganti 5 -CAG AAA CTG TTC TAA TAA TAG CG complementary to positions 6-29 of SL RNA TBNT CGC AAC ATG CAT GCA TCC TTC TTG complementary to the TbNT8.1 coding region TBNT81 anti 5 - TCG ATG CCT TGA TGA TGC AC complementary to the TbNT8.1 coding region Srp54 5'-CGT CTA GAA CGT GAA GGA GTT TGT AAA T complementary to positions from 80 to 99 of the SRP54 coding region, including a XbaI site Srp54 anti 5'-CCC AAG CTT TCT CTT CGA ACA GTG CCG AT complementary to positions from 594 to 613 of the SRP54 coding region, including a HindIII site Sirt6 5 -CAG TCT CGA GGT CCC ACA GTG CGT GG complementary to the promoter region of the Sirt6 gene from human Sirt6 anti 5 -CAG TAG ATC TCT GTC CGG CTC TGT CC complementary to the promoter region of the Sirt6 gene from human Pex 5 - CTC CCC GAG TGG ACC AGG complementary to the stuffer region of the SRα-hp-1 and 2 constructs Pex anti 5 - TCT AGA GCG GCG CTT AAA complementary to the stuffer region of the SRα-hp-1 and 2 constructs '- TCC TGG AAG CCG CGC GTC GC complementary to positions of 18S rrna

4 Figure S-1 SRα depletion does not affect mrna stability. A. Uninduced (-Tet) and induced cells (+Tet) 3 days after tetracycline addition were treated with 20 µg/ml actinomycin D for 0 to 90 minutes as indicated. RNA was prepared from the treated cells and subjected to Northern analysis with the indicated probes. rrna hybridization was used to control RNA loading. B. The hybridized signals were measured by densitometry, and the level of mrnas was calibrated using the hybridization of 28 S rrna present in the samples. The decay curves of TbNT8.1, fibrillarin and SRP54 mrna are shown in panels 1, 2 and 3, respectively. Half-life of the respective mrna is indicated in the graphs. Figure S-2 In vivo labeling of cells depleted of SRα. Uninduced (-Tet) and induced cells (+Tet) on the third day after induction were labeled with [ 35 S]-methionine/cysteine mixture. Pulse was for 5-min followed by 15-min chase. Proteins were fractionated on a 10% (w/v) SDS-polyacrylamide gel. The gel was stained with coomassie blue (as a control for loading), and subjected to autoradiography. Figure S-3: SL RNA and mrna reduction upon tsnap42 silencing. A. RNA was prepared from uninduced cells and cells depleted of tsnap42 for 3 days and subjected to Northern analysis with SL RNA probe and 5.8 S rrna probe (used as a control for equal loading). B. The same as in A, but using mrna probes as indicated. Figure S-4: SLS is induced in SRα silenced cells. A. SL RNA level in SRα depleted cells. RNA was prepared from transgenic parasite expressing a hairpin RNA to silence the SRα gene. The silencing construct is specific from nt 38 to 490 of the SRα gene open reading frame. Total RNA (20 µg) from Induced (+Tet) and uninduced (-Tet) cells 3 days after induction was subjected to Northern analysis with SL RNA and 5.8S rrna random-labeled probes. B. tsnap42 localization and accumulation in SRα depleted cells. Uninduced cells (-Tet), and silenced cells (+Tet) on the third day after induction were fixed with 4% (v/v) formaldehyde for 25 min, incubated with anti-tsnap42 (indicated by

5 arrows) antibody and detected by FITC-conjugated second antibody. The nucleus was stained with DAPI. Scale bar, 5 μm.

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