METTL3 Dysregulates RNA Splicing by Translational Control of Splicing Factors via m 6A Modification in CLL
Authors: Wu Yiming , Jin Meiling , Fernandez Mike , Hart Kevyn , Liao Aijun , Fernandes Stacey M. , McDonald Tinisha , Ghoda Lucy , Roeth Daniel , Kalkum Markus , Pillai Raju , Chen Jianjun , Brown Jennifer R , Danilov Alexey V. , Rosen Steven T. , Siddiqi Tanya , Wang Lili 
Published: November 2021

Abstract
RNA splicing dysregulation is a hallmark of chronic lymphocytic leukemia (CLL). Although somatic mutations in SF3B1 or U1 snRNA present in &gt;20% of CLL patients, general splicing defects cannot be fully explained by genetic alterations of spliceosome alone. We reported that splicing factors are upregulated at protein, but not RNA, level in CLL compared to normal B cells by an integrated transcriptomic and proteomic analysis. This highlights a post-transcriptional layer of regulation that controls the abundance of splicing factors and contributes to RNA splicing dysregulation in CLL, with mechanism that has yet to be elucidated.
To discover these regulators, we performed weighted correlation network analysis and found expression of many splicing factors strongly correlated with the abundance of METTL3. METTL3 is an RNA methyltransferase that modifies N 6-methyladenosine (m 6A) on mRNA and regulates the translation of m 6A-installed transcripts. Primary CLL cells have consistent upregulated protein expression of METTL3 and exhibit an increased m 6A level on mRNA. To identify m 6A modification sites in normal and CLL B cells, we performed m 6A sensitive RNase sequencing and found transcripts with differential m 6A modification are highly enriched in the RNA splicing pathway (q=1.8E-4). Consistent with this, these transcripts appear to have a higher m 6A density. These results raised up a possibility that METTL3 translationally controls the expression of splicing factors through m 6A modification.
To examine whether METTL3 impacts splicing factor expression, we performed an integrated ribosome profiling (Ribo-seq) and RNA sequencing using CLL cell line HG3 with or without METTL3. Knockout (KO) of METTL3 decreased overall translation efficiency with RNA splicing as the most significantly affected pathway (q=2.1E-52). We further confirmed that KO of METTL3 or treatment with METTL3 inhibitor STM2457 decreases the expression of many splicing factors in HG3 cells. However, we detected no protein changes in known METTL3 targets (BCL2, MYC), highlighting splicing factors as the preferred targets of METTL3 in CLL. Moreover, overexpression of wildtype but not catalytic mutant METTL3 restored splicing factor expression defects in METTL3 KO cells, demonstrating that the regulation of splicing factor expression is methyltransferase activity dependent.
To dissect the mechanism of how METTL3 preferentially influences the expression of splicing factors via m 6A modification, we mapped out METTL3 responding m 6A sites using methylated RNA immunoprecipitation sequencing (MeRIP-seq). Consistent with its role as an methyltransferase, we detected 5875 hypomethylated and 1409 hypermethylated sites upon METTL3 KO. Most downregulated splicing factors harbored hypomethylation around the stop codon region, including SF3B1, SF3A2 and SR proteins, indicating they are direct targets of METTL3. To validate this association, we utilized m 6A editing platform dCasRx-METTL3 to install m 6A at the stop codon region of endogenous SF3B1 transcripts, leading to increased SF3B1 protein expression. These results provided evidence that METTL3 directly regulates splicing factor protein expression via m 6A mediated translational control. Unexpectedly, we also discovered that hypermethylated transcripts are highly enriched in RNA splicing with most affected sites localized at the CDS regions. The hypermethylation of these transcripts can be caused by a combined upregulation of m 6A writer (METTL16) and eraser (ALKBH5) protein upon KO of METTL3. To determine how hypermethylation affects expression of splicing factors, we conducted an integrated MeRIP-seq and Ribo-seq analysis and observed a convergence of increased ribosomal density and hypermethylated adenosines. Harnessing dCasRx-METTL3 platform, we installed m 6A at the CDS region of SF3A3 and confirmed the downregulation of protein by immunoblot. This implicates that, as an alternative mechanism, METTL3 regulates the translation of splicing factors via m 6A mediated decoding process.
Altogether, our results uncovered a novel regulatory axis of METTL3 as a regulator for splicing dysregulation in CLL. We propose that METTL3 regulates splicing factor expression through m 6A-mediated translational control. Our study highlights a post-transcriptional layer of m 6A modification as a major contributor to genetic lesion-independent splicing defects in CLL.

Disclosures
Brown: Gilead, Loxo/Lilly, SecuraBio, Sun, TG Therapeutics: Research Funding; Invectys: Other: Data Safety Monitoring Committee Service; Abbvie, Acerta/Astra-Zeneca, Beigene, Bristol-Myers Squibb/Juno/Celgene, Catapult, Eli Lilly, Genentech/Roche, Janssen, MEI Pharma, Morphosys AG, Nextcea, Novartis, Pfizer, Rigel: Consultancy. Danilov: Abbvie: Consultancy, Honoraria; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy, Research Funding; Beigene: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Gilead Sciences: Research Funding; Rigel Pharm: Honoraria; Genentech: Consultancy, Honoraria, Research Funding; Bayer Oncology: Consultancy, Honoraria, Research Funding; SecuraBio: Research Funding; Astra Zeneca: Consultancy, Honoraria, Research Funding; Bristol-Meyers-Squibb: Honoraria, Research Funding. Siddiqi: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Speakers Bureau; Kite Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Oncternal: Research Funding; TG Therapeutics: Research Funding.