Splicing inhibition by a single methyl group
In humans, the majority of N6-methyladenosine (m6A) is deposited by the heterodimer METTL3/METTL14. However, a subset of marks is placed by the writer enzyme METTL16. The Pillai lab previously gained insights into the structure and function of this writer in mammalian organisms (Mendel, Chen et al., 2018).
To further study this writer, they turned to the METTL16 homolog in C. elegans called METT-10. C. elegans is an interesting model to study, as in contrast to mammals, it lacks a METTL3/METTL14 homologue. For the worm research, the Pillai lab joined forces with a lab headed by another Schümperli group alumnus Florian Steiner (both at the University of Geneva). Little did they know at the beginning, that this research would lead them into the RNA splicing field, discovering a novel mechanism for its regulation.
As a start, they mapped the m6A transcriptome of C. elegans. Despite the similar amount of m6A in C. elegans and mouse, they identified only 176 m6A peaks in worms, while a similar analysis in mouse identified over 20 000 peaks. Upon knockout of the m6A methylase METT-10, they observed that the m6A methylation levels went down for several transcripts, including sites located in the transcripts of U6 snRNA and sams-3, -4 and -5, which are SAM synthetase transcripts. SAM is the most important methyl donor for methylation reactions in the cell, including m6A deposition. The identification of these sites mimics the situation in mammals, in which U6 snRNA and the SAM synthetase MAT2A are targets of METTL16.
However, the location of the methylation sites in the SAM synthetase transcripts differs in mice and worms. While the mouse Mat2a has six methylation sites in the 3’-UTR, the transcripts of C. elegans sams-3, -4 and 5 are methylated at a single site, which is the 3’ splice site AG of the intron 2 in the sams pre-mRNA transcripts. Methylation of the 3’ splice site leads to splicing inhibition of the corresponding intron and to reduced levels of the SAM synthetase. Through further experiments, they could show that the m6A mark at the 3’ splice site prevents binding of the essential splicing factor U2AF35, inhibiting splicing.
The researchers could further show that the methylation of 3’ splice sites only occurs under high nutrient conditions. Under low nutrient conditions, the methylation mark is absent and splicing produces mature transcripts. “The connection to nutrient levels seems obvious, but we discovered it initially by chance” says Ramesh Pillai, last author of the study. When performing the final round of biological replicates, the worms were grown accidentally on low-nutrient plates and the methylation at the 3’ splice sites disappeared. “At first, this came as a shock because we thought our initial experiments and model were wrong. But then, we went with Kamila Delaney (PhD student in the Steiner lab) through the lab journal entries, and we noticed the usage of different plates.” recalls Mateusz Mendel, first author of the paper and PhD student in the Pillai lab.
In conclusion, they could show that both C. elegans and mammals use a mechanism depending on the homologous m6A methylases METT-10 and METTL16, respectively, targeting SAM synthetase transcripts to regulate SAM levels in response to nutrition. However, the underlying regulation mechanism in the two systems is different. In response to high SAM levels, worms downregulate SAM synthetase levels through METT-10-mediated 3’ splice sites methylation, leading to splicing inhibition. In mammals, it was previously shown, that METTL16 promotes the splicing of the Mat2a transcript when SAM levels are low. However, this is not through its catalytic activity, but through vertebrate-specific regions located in its C-terminal half. When SAM levels are high, mammalian METTL16 methylates its Mat2a binding sites and dissociates from the transcript, leading to loss of splicing promotion. This methylation sites further fine-tune Mat2a levels through recruitment of an m6A reader YTHDC1 to promote degradation of the Mat2a transcript.
The big question then was if methylation of 3’ splice sites can control splicing in mammals. Through experiments with splicing reporter constructs in vivo using HeLa cells, and in vitro with HeLa S3 extracts, the researchers could demonstrate that m6A methylation of the 3’ splice site can inhibit the human splicing machinery. Moving beyond the reporter constructs, computational analysis revealed the existence of around 1000 splice sites in the mouse transcriptome that could potentially be regulated by METTL16. In an in vitro methylation assay, the majority of the top ten most promising sites could be methylated by METTL16. Moreover, through sequencing the transcriptome of Mettl16 knock out mouse embryos, they identified two splice sites, whose use is upregulated in the absence of METTL16. These results do hint, but not finally prove, that these sites are regulated directly by METTL16. The findings of the Pillai and Steiner labs have just been published in Cell (Mendel et al., 2021). Further research could help to clarify if 3’ splice site methylation by METTL16 is a splicing regulatory mechanism also conserved in mammals.
Mendel et al. (2021) Cell 184(12), 3125-3142.e25 (Open Access)
Mendel, Chen et al. (2018) Molecular Cell 71(6), 986-1000.e11 (Open Access)
Text: Dominik Theler