Roles of RNA methylation writers in gene expression and cancer

After a relatively slow start, the field of RNA epigenetics has exploded in recent years. As with DNA, RNA modifications are controlled by proteins capable of post-transcriptionally writing, reading, or erasing position-specific modifications to affect downstream processes. In this dissertation, we focus on the molecular mechanisms surrounding RNA modification writers and how they contribute to human cancer. Our studies center upon BCDIN3D, a Bin3 domain-containing RNA methyltransferase. A 2012 study (Xhemalçe et al.) established BCDIN3D catalytic activity on the 5’ mono-phosphate of specific precursor miRNAs while intriguingly showing that downregulation of BCDIN3D in human triple negative breast cancer cell line MDA-MB-231 led to decreased transformation and invasion in vitro. In order to understand the mechanisms underlying these effects on human cancer cells, we performed in depth analyses of the RNA and protein interactome of BCDIN3D. Sequencing of BCDIN3D-associated RNAs through TGIRT-seq revealed a dominant tRNA [superscript His] interaction and a new role for BCDIN3D in regulation of miR-4454, which is possibly derived from noncanonical processing of the tRNA [superscript His] 3’ end. Proteomic analysis of BCDIN3D interactors unexpectedly revealed the presence of two 5-methylcytosine (m⁵C) RNA methyltransferases, NSUN2 and NSUN5. While the interaction with NSUN2 is likely related to tRNA [superscript His] m⁵C modification, characterization of NSUN5 unearthed a number of intriguing new putative targets and pathways influenced by m⁵C activity, as well as promise for future therapeutic development in human cancers.Cellular and Molecular Biolog

Search for Novel DNA Modifications in Saccharomyces cerevisiae mtDNA using Single Molecule Real Time Sequencing and Effects of Mitochondrial Metabolic Dynamics on Gene Expression

In the past five years, Single Molecule Real Time (SMRT) sequencing technology has been found to be a reliable indicator of certain epigenetic modifications in bacterial genomes. The genome of the model organism Saccharomyces cerevisiae has long been thought to be free of DNA level modification, but literature surrounding this subject is conflicting. Additionally, the mitochondria of S. cerevisiae control the transition between three distinct chronological life phases – exponential, postdiauxic, and stationary - as defined by their main metabolic processes. This study attempted to identify base modifications to mtDNA using PacBio sequencing while additionally establishing gene expression changes as a result of altered mitochondrial metabolic capabilities. PacBio results showed intriguing results but statistical analysis proved experimentation with improved protocols were necessary. Multiple genes with unknown or uncharacterized function were also shown to have significant differential expression between metabolic life phases

An Epigenetic Trap Involved in Olfactory Receptor Gene Choice

Reporting recently in Cell, Lyons et al. (2013) reveal key roles for transient LSD1 histone demethylase activity in activation of a single olfactory receptor allele and suppression of the rest of the olfactory receptor gene family, thereby locking in the expression of a single olfactory receptor per sensory neuron

Who Watches the Watchmen: Roles of RNA Modifications in the RNA Interference Pathway.

RNA levels are widely thought to be predictive of RNA function. However, the existence of more than a hundred chemically distinct modifications of RNA alone is a major indication that these moieties may impart distinct functions to subgroups of RNA molecules that share a primary sequence but display distinct RNA "epigenetic" marks. RNAs can be modified on many sites, including 5' and 3' ends, the sugar phosphate backbone, or internal bases, which collectively provide many opportunities for posttranscriptional regulation through a variety of mechanisms. Here, we will focus on how modifications on messenger and microRNAs may affect the process of RNA interference in mammalian cells. We believe that taking RNA modifications into account will not only advance our understanding of this crucial pathway in disease and cancer but will also open the path to exploiting the enzymes that "write" and "erase" them as targets for therapeutic drug development

Model for the mode of action of the BCDIN3D RNA methyltransferase on the biogenesis of specific miRNAs.

<p>BCDIN3D’s enzymatic activity consists in the methylation of the two available oxygen moieties of the 5′ monophosphate, which removes the 5′ monophosphate charge and makes it bulkier. This methylation blocks the processing of specific miRNAs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref008" target="_blank">8</a>], possibly through perturbing the interaction of the 5′ monophosphate with its binding pocket in Dicer [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref008" target="_blank">8</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref014" target="_blank">14</a>].</p

List of known messenger or miRNA modifications, with chemical structures, abbreviation, writers, erasers, and readers.

<p><b>*</b>The <i>Drosophila</i> dTET enzyme has recently been shown to hydroxylate 5meC on RNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref011" target="_blank">11</a>].</p

Model for how m6A may stimulate pri-miRNA processing.

<p>m6A is deposited on pri-miRNA by METTL3 and is thought to stimulate the recruitment of Drosha/DGCR8 for co-transcriptional processing of pri-miRNA to pre-miRNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006139#pgen.1006139.ref027" target="_blank">27</a>]. The question mark is to highlight that the identity of the full set of m6A readers in pri-miRNAs is unknown. In yellow is shown RNAP II on DNA surrounded by nucleosomes.</p

Simplified schematic of (A) the miRNA biogenesis pathway and (B) the interaction between the mRNA and miRNA.

<p><b>(A)</b> The primary miRNA precursor (pri-miRNA) is synthesized by RNAP II. The pri-miRNA is first cleaved by Drosha to release a hairpin loop–shaped RNA called pre-miRNA. The loop of this pre-miRNA is further cleaved by Dicer to generate a miRNA duplex. The miRNA duplex is dissociated and the passenger strand (dashed line) is discarded while the guide strand is loaded onto the Argonaute protein to form an active RISC complex. <b>(B)</b> Example of miRNA–target mRNA interaction by base-pairing mainly at the seed region of miRNA (nt 2–8) but also on other downstream regions of the miRNA. In the mRNA, the coding region is represented as a double line. <b>N.B.</b> The asterisks indicate the m7G cap (7-methyl-guanosine with 5′, 5′-triphosphate linkage).</p

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-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