Figure 3.

IGHMBP2 associates with small RNAs and in particular with tRNA-Tyr. (A) Lysates from mouse MN1 cells stably expressing FLAG-IGHMBP2 or vector only (FLAG-vector) were probed on a western blot with a monoclonal antibody against the FLAG epitope. (B) RNA immunoprecipitations (RNA-IP) were performed with anti-FLAG antibody from FLAG–IGHMBP2-expressing or mock-transfected (vector only) MN1 cells. RNA was isolated from the immunoprecipitates, 3′-end-labeled with [5′-³²P]-pCp and T4 RNA ligase and resolved by electrophoresis on a 10% denaturing PAGE. Nucleotide sizes of the radiolabeled marker (M) are shown on the left. Red arrow at ∼40 nt indicates small RNAs that were directionally cloned and sequenced. (C) Secondary structure of mouse tRNA-Tyr (tRNA-882; numbering is adapted from 13) with cloned sequences shown in red, including the post-transcriptionally added CCA trinucleotide at the 3′-end. Nucleotide differences of tRNA 880 (tRNA-Tyr2) are indicated in green; all other nucleotides between the various tRNAs-Tyr listed above are identical. The pseudo-uridine modification of uridine present in the center of the anticodon of all tRNA-Tyr is shown with the symbol ψ. (D) Sequences of mouse tRNA-Tyr genes contained in the modifier locus (see Fig. 4). Introns are in lower case; note that intronic sequences differ between the various tRNA-Tyr genes. (E) RNA-IP was performed with 11-24 mAb against endogenous IGHMBP2 from human HeLa cells or with non-immune mouse serum (NMS, negative control). RNA was isolated from the immunoprecipitates, 3′-end-labeled with [5′-³²P]-pCp and T4 RNA ligase and resolved by electrophoresis on a 10% denaturing PAGE. Nucleotide sizes of the radiolabeled marker (M) are shown on the left. Red arrow indicates tRNAs. (F) RNA-IP was performed from HeLa cells as in (E), and the isolated RNA was transferred to a nylon membrane and probed with a 5′-end-labeled DNA oligo, antisense to tRNA-Tyr.