Date Published: July 22, 2019
Publisher: Public Library of Science
Author(s): Calder W. Reinsborough, Hélène Ipas, Nathan S. Abell, Ryan M. Nottingham, Jun Yao, Sravan K. Devanathan, Samantha B. Shelton, Alan M. Lambowitz, Blerta Xhemalçe, Anita K. Hopper
Abstract: 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-4454 is not synthesized from its 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.
Partial Text: The 5’ ends of eukaryotic RNAs are vital for determining their processing and function . The most well-known 5’ end RNA modification is the m7G cap, which has multiple functions, including of protecting messenger RNAs from exonucleolytic degradation, and of marking them for nucleo-cytoplasmic export and translation . Additionally, a chemically simpler 5’ end modification by O-methylation occurs directly on 5’ phosphate ends, either on the γ-phosphate of nascent tri-phosphorylated snRNAs, or on the α-phosphate of processed monophosphate RNAs . These methylations are carried out by the Bicoid Interacting 3 (BIN3)  family of methyltransferases, which are conserved from fission yeast to humans . In humans, two BIN3 related enzymes, MePCE and BCDIN3D, have been identified. Jeronimo et al., uncovered that MePCE methylates the γ-phosphate of the 7SK snRNA , while we discovered that BCDIN3D methylates the 5’ monophosphate of two specific microRNA precursors, pre-miR-145 and pre-miR-23b, both in vitro and in cells, to inhibit their processing into mature miRNA by Dicer . Our initial analysis in MCF-7 cells also suggested that other microRNAs could be methylated by BCDIN3D . However, due to lack of methods to specifically enrich 5’ phospho-methylated RNAs, the methylation of other types of RNAs with 5’ monophosphates was not analyzed in our initial study.
Based on our results, we hypothesize that BCDIN3D forms an RNP with mature tRNAHis that is phospho-methylated (Figs 1 and 2). This could result from relatively slow product release as recently shown for the MePCE phosphomethyltransferase . Stable interaction with tRNAHis may regulate BCDIN3D activity towards its other targets, including precursor miRNAs  or other yet to be uncovered phospho-methylated RNAs, by affecting BCDIN3D structure and/or RNA target selection. In this context, it will be of particular interest to investigate which cellular conditions disrupt BCDIN3D interaction with tRNAHis, and how in turn those conditions affect BCDIN3D methyltransferase activity towards its other RNA targets. The gel shift assay for probing tRNAHis phospho-methylation status developed here (Fig 2) provides a fast tool for testing tRNAHis methylation in cells (S6 Fig), and iterations of this assay could be used for other BCDIN3D targets as well.