MicroRNAs (miRNAs) are small-noncoding RNAs that regulate gene expression by binding with imperfect complementarity to target mRNAs. miRNA expression levels are altered in many types of cancer and other diseases and this has led to great interest in its biogenesis. While mature miRNAs are composed of 21-25 nucleotides, they begin as much longer transcripts, known as primary miRNAs (pri-miRNAs). Prior to exiting the nucleus and becoming a mature miRNA, each pri-miRNA must be processed to a pre-miRNA hairpin. While it is known that the cleavage is imparted by a complex composed of the RNA binding protein DGCR8 and the type III RNase Drosha, the mechanism by which the Drosha-DGCR8 complex recognizes the correct region for cleavage has been under investigation.
In an effort to understand this process, Alarcón et al. set forth to determine how pri-miRNAs are marked for cleavage at the junction of the stem and flanking single-stranded RNA region. They reasoned that the RNA is most likely marked through a post-transcriptional modification and searched for a common sequence motif in miRNA encoding genomic regions. They observed an over-representation of GGAC, which is a recognition sequence for the RNA methyltransferase-like 3 (METTL3). METTL3 is responsible for the methylation of adenosine at position 6 (m6A). Through RNA sequencing, they found that the m6A modification was abundant in pri-miRNA transcripts and that m6A was located within the canonical METTL3 motifs. Upon METTL3 depletion, the authors found that ~70% of mature miRNAs were downregulated by at least 30%.
Next, the group performed a series of expression studies and found that deletion of METTL3 led to a decrease in m6A modified pri-miRNAs and fewer processed miRNAs. Conversely, they observed that overexpression of METTL3 was sufficient to increase mature mRNAs. To directly show that METTL3 was responsible for m6A methylation in vivo, Alarcón and colleagues performed high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). Using an antibody against endogenous METTL3, they revealed a significant enrichment of METTL3 within the canonical METTL3 motif. They also found that this motif is highly conserved across the 100 vertebrate genomes they investigated.
The authors then wanted to test the direct role of the m6A modification in pri-miRNA processing. To do this, they artificially added m6A to pri-miRNA through in vitro transcription with N6-methyladenosine-5'-triphosphate and looked at pri-miRNA processing. They observed that the m6A modified pri-miRNAs were more efficiently processed than pri-miRNAs that were mutated to eliminate m6A sites.
In the next set of experiments, Alarcón and colleagues focused on how these elements interact. First, through immunoprecipitation experiments, they determined DGCR8 interacts with METTL3 through RNA. They also demonstrated that DGCR8 interacts with the methylation sites in vivo. The authors conclude that this methylation system may act to confer specificity to distinguish between pri-miRNAs and mRNAs, which also contain similar secondary structures.
In summary, this research demonstrates that the methylation of adenosine through METTL3 is an important marker for pri-miRNA processing. This finding is particularly interesting because similar to miRNAs, altered METTL3 expression is documented in multiple forms of cancer. This research provides a connection between these two cancer markers for the first time.
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