FIG. 2.

Identification and characterization of SLA1 cross-linked species in T. brucei. (A) Identification of SLA1, SL RNA, and U5 cross-linked species. SmD1 cells were grown with or without tetracycline (Tet + or −, respectively). AMT was added to cells 40 h after induction. Cells were either UV cross-linked for 30 min (365 nm) (UV +) or left untreated (UV −). RNA was extracted and subjected to splint labeling using oligonucleotide 6211, specific to SLA1 (lanes 1 to 4); oligonucleotide 84752, specific to SL RNA (lanes 5 and 6); oligonucleotide 13290, specific to SL RNA from SmD1 cells (lanes 7 and 8); or oligonucleotide 55448, specific to U5 snRNA (lanes 9 and 10). The reaction products were separated on a denaturing 6% polyacrylamide gel alongside a labeled pBR322/HpaII digest marker. The identities of the splint-labeled RNAs are indicated on the right. Nonspecific labeling is marked by an asterisk. (B) Cellular fractionation to determine the localization of SLA1/SL RNA cross-linked species. UV cross-linking was performed with SmD1 cells as described for panel A. Nuclear (Nuc, N) and cytoplasmic (Cyt, C) fractions were prepared from the cells as described in Materials and Methods, and RNA was subjected to splint labeling using oligonucleotide 6211, specific to the SLA1 3′ end (lanes 1 to 4); oligonucleotide 84752, specific to the SL RNA 3′ end (lanes 5 and 6); or oligonucleotide 13290, specific to the SL RNA extended 3′ end (lanes 7 and 8). The same RNA was subjected to primer extension using oligonucleotides complementary to 7SL RNA (1097), Tb11Cs2C2 (36591), and U6 snRNA (12407). The reaction products were separated on a denaturing 6% polyacrylamide gel alongside a labeled pBR322 marker. The values at left indicate the RNA sizes (in nucleotides). (C) Identification of SL RNA cross-linked species using oligonucleotide-directed RNase H cleavage. Splint-labeled SL RNA cross-linked species and splint-labeled SL RNA (as shown in panel A) from uninduced cells (lanes 1 to 6) and SmD1 induced cells (lanes 7 to 9) were excised from the gel and subjected to RNase H cleavage with an antisense oligonucleotide (Oligo) specific to SL RNA (lanes 2, 4, and 8), SLA1 (lanes 5 and 9), or U5 snRNAs (lane 6) or with no oligonucleotide as a control (lanes 1, 3, and 7). Reaction products were separated on a denaturing 6% polyacrylamide gel. The cross-linked species and their cleavage derivatives are marked with dashed lines, and their identities are indicated. (D) Identification of SLA1 cross-linked species using oligonucleotide-directed RNase H cleavage. The experiment was the same as that described for panel C, but the RNA was splint labeled with an SLA1-specific probe and the cross-linked species was cleaved with an antisense oligonucleotide specific to SLA1 (lanes 2 and 5) or SL RNA (lane 4) or with no oligonucleotide as a control (lanes 1 and 3).