Supplementary Table 1: Oligonucleotides and Plasmids 913954 5'- GCT CTA GAG AAC TTG AAG TAC AGA CTG C 913955 5'- CCC AAG CTT ACA GTG TGG CCA TTC TGC TG 223396 5'- 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 AD306 5 -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. AD307 5 - 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. 953610 5'- CCG CTC GAG AGC CGG AGC GCA TTG CTC TG complementary to positions 1-16 of 7SL RNA 953611 5'- CCC AAG CTT CCG CCT CGC GAC GAC ACT TG complementary to positions 253-278 of 7SL RNA
12407 5'- TTC GAG AGA TAT AGC T complementary to positions 81-96 of U6 TB-U3 5'- GTT CGA TGA ACG GCA complementary to positions 105-120 of U3 162920 5'- TCT CCT CAC CCC CTC GCG complementary to positions 3-20 of tubulin 162921 5'- ATG CAG ATA GCC TCA CGC complementary to positions of 123-141 tubulin 4353 5'- GTA GCG ATG CTG ACG TGC AA complementary to positions 1148-1167 of 28S rrna 5262 5'- CAC TCA CAC ACA CAT GGC TAT complementary to positions 2066-2085 of 28S rrna 21113 5'- AAC TAA CGC TAT TAT TA complementary to positions 1-17 of SL RNA 9091 5'- GCA GGA ACC AAC AGC ACA ATG CG complementary to positions 90-110 of SL RNA Sm03 5'- CCA TCC CCG TGC ATG CCA CAT TTC TCA GTG TCG complementary to positions 90-110 of 5.8S rrna 1222B12 5'- CCG CTC GAG TAG GGA GCA TCC ACA TGA A complementary to positions 389-407 of fibrillarin 1222D12 5'CCC AAG CTT GTT GTA CTG CTT TCT TCA TA complementary to positions 879-898 of fibrillarin 3083 5'- GAT CAC TGC CGG TGG TAT CT complementary to positions 1326-1345 of vhppase 3084 5'- GCA CCG AAA TGT AAC CGA GT complementary to positions 1686-1705 of vhppase 3081 5'- ATT TTT CCA TCG GCT TTG TTG 3082 5'- ACC CAA AAC TCA TTC GTT GC 3079 5'- TCC CTT TAT CTG CTC GCT GT complementary to positions 154-172 of hexose transporter complementary to positions of 514-533 hexose transporter complementary to positions 44-63 of EP 3080 5'- TAG AAT GCG GCA ACG AGA C complementary to positions 420-438 of EP
1106 5'- CTT AGC CAT GCA TGC CTC complementary to positions 1106-1132 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 97-115 of srrna4 Sh3-sl-neg 5 -CCA TAA CGG CTT AAG CAC AAG complementary to positions -132 - -111 upstream of SL RNA Sh4-sl-neganti 5 -CAG AAA CTG TTC TAA TAA TAG CG complementary to positions 6-29 of SL RNA TBNT81 5 - 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 1115 5'- TCC TGG AAG CCG CGC GTC GC complementary to positions 1115-1134 of 18S rrna
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
arrows) antibody and detected by FITC-conjugated second antibody. The nucleus was stained with DAPI. Scale bar, 5 μm.
Figure S-1: Figure S-2:
Figure S-3: Figure S-4: