Limited effects of m6A modification on mRNA partitioning into stress granules

Abstract

The presence of the m6A modification in mammalian mRNAs is proposed to promote mRNA recruitment to stress granules through the interaction with YTHDF proteins. We test this possibility by examining the accumulation of mRNAs in stress granules in both WT and ∆METTL3 mES cells, which are deficient in m6A modification. A critical observation is that all m6A modified mRNAs partition similarly into stress granules in both wild-type and m6A-deficient cells by single-molecule FISH. Moreover, multiple linear regression analysis indicates m6A modification explains only 6% of the variance in stress granule localization when controlled for length. Finally, the artificial tethering of 25 YTHDF proteins on reporter mRNAs leads to only a modest increase in mRNA partitioning to stress granules. Since most mammalian mRNAs have 4 or fewer m6A sites, and those sites are not fully modified, this argues m6A modifications are unlikely to play a significant role in recruiting mRNAs to stress granules. Taken together, these observations argue that m6A modifications play a minimal, if any, role in mRNA partitioning into stress granules.

A New Phase of Networking: The Molecular Composition and Regulatory Dynamics of Mammalian Stress Granules

“…The SG transcriptome is highly enriched for m6A-modified mRNAs relative to total cellular mRNA, with a strong correlation between the extent of m6A modification and partitioning to SGs. ,, However, >97% of transcripts contain four or fewer m6A sites, and these sites are modified at rates of less than 100% . More recent bioinformatic analyses suggest that mRNA length has a stronger impact on SG partitioning than the presence of m6A sites . In terms of mechanism, m6A modifications are catalyzed by a writer complex comprising METTL3, METTL14, and WTAP and are recognized by the YTH domain-containing proteins YTHDF1/2/3, which variably colocalize with SGs following stress treatments. ,,,, It should be noted that genetic depletion of writer complex components significantly reduces the accumulation of m6A-modified mRNAs in SGs, although SG formation is not impaired. , While these data are based on the detection of mRNAs in bulk, a more quantitative evaluation of 11 different mRNAs did not reveal any significant differences in SG partitioning between wild-type cells and a mettl3 knockout .…”

“…In terms of mechanism, m6A modifications are catalyzed by a writer complex comprising METTL3, METTL14, and WTAP and are recognized by the YTH domain-containing proteins YTHDF1/2/3, which variably colocalize with SGs following stress treatments. ,,,, It should be noted that genetic depletion of writer complex components significantly reduces the accumulation of m6A-modified mRNAs in SGs, although SG formation is not impaired. , While these data are based on the detection of mRNAs in bulk, a more quantitative evaluation of 11 different mRNAs did not reveal any significant differences in SG partitioning between wild-type cells and a mettl3 knockout . Variable observations have also been obtained from analyses of YTHDF genetic knockdowns, ranging from a significant impairment of SG localization by m6A-modified mRNAs to no detectable effect, potentially due to differences in experimental approaches. , Overall, Khong et al posit that RNA targeting to SGs results from the cumulative contribution of multiple factors and that m6A modification plays a relatively minor role. Interestingly, SG RBPs such as G3BP1/2, RBM42, CAPRIN1, and USP10 preferentially bind unmethylated sequences over m6A-modified RNAs, , suggesting distinct mechanisms for methylation-dependent and -independent transcript recruitment into SGs.…”

Emerging Implications of Phase Separation in Cancer

“…[165,166] An argue study indicates that mRNA length, rather than m 6 A modifications, has a promotion effect on mRNA partitioning in SGs. [167] Additionally, m 6 A modifications were shown to repress RNA binding to G3BP1/2, ubiquitin-specific protease 10 (USP10), Caprin-1, and RNA-binding motif 4 (RBM4) which are linked to the formation of SGs. [168] Thus, the inconsistent observations above implicate that the role of m6A in SGs formation is still a debatable issue.…”

Stress granules dynamics: benefits in cancer

“…These findings collectively suggest that m 6 A modification might modulate SGs targeting of RNAs. However, a recent study has found that m 6 A modifications have limited effects on mRNA recruited into SGs ( 39 ). Thus, the relationship of m 6 A modification with SGs targeting remains to be elucidated.…”