BCDIN3D regulates tRNAHis 3’ fragment processing

5’ ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5’ monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4455 is not synthesized from the annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3’ end of tRNAHis. Moreover, we were able to generate this “miRNA” in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5’P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3’-fragment/”miR-4454” levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3’-fragment processing without negatively affecting tRNAHis’s canonical function of aminoacylation. In order to purify RNAs interacting with BCDIN3D, we used HeLa-S3-FlpIn cells containing a single insertion at a FRT locus of BCDIN3D tagged with a FLAG tag at its C-terminus (BCDIN3Df). We first purified BCDIN3Df with a protocol that combines FLAG antibody co-immunoprecipitation, followed by FLAG peptide elution. We then extracted RNAs from FLAG eluates of Control and BCDIN3Df cells with a protocol that allows recovery of RNAs of all sizes. Finally, we used a TGIRT-seq protocol that combines template switching to the RNA 3’ end and a highly processive Thermostable Group II Intron Reverse Transcriptase for cDNA synthesis.