October 2022
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272 Citations
Cell
The discovery of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway arose from investigations of how cells respond to interferons (IFNs), revealing a paradigm in cell signaling conserved from slime molds to mammals. These discoveries revealed mechanisms underlying rapid gene expression mediated by a wide variety of extracellular polypeptides including cytokines, interleukins, and related factors. This knowledge has provided numerous insights into human disease, from immune deficiencies to cancer, and was rapidly translated to new drugs for autoimmune, allergic, and infectious diseases, including COVID-19. Despite these advances, major challenges and opportunities remain.
![Figure 2. Each individual m 6 A modification is modified mostly with the same level for each of the three cell fractions. (A) Comparison of individual m 6 A peak signal strength in CA-RNA and nucleoplasmic RNA for the same m 6 A peaks (each dot is an individual m 6 A peak: no changes in gray, CA-RNA higher in orange, and nucleoplasm higher in dark blue). FDR <5%, Fisher's exact test. To determine m 6 A peaks that are higher in CARNA, for each m 6 A peak region, we enumerated reads of m 6 A immunoprecipitation and the input for CA-RNA and Nucleoplasm RNA, evaluating statistical significance with Fisher's exact test, using stringent FDR cutoffs to correct for multiple hypothesis testing. The determination that an m 6 A peak region was higher in CA-RNA required (1) the reads of mRNAs in m 6 A peak regions to be adequate for m 6 A peak region detection in both CA-RNA and nucleoplasmic mRNA (reads per kilobase per million mapped reads [RPKM] ≥1) and (2) FDR ≤0.05 and a twofold or higher of peak region enrichment in CA-RNA compared with nucleoplasmic mRNA. At a lower cutoff (e.g., ≥1.5-fold), the same conclusion held: that most m 6 A peaks are modified with the same level between CA-RNA and nucleoplasmic mRNA. (B) Comparison of individual m 6 A peak signal strength in nucleoplasmic RNA to cytoplasmic RNA for the same m 6 A peak. The same statistic criteria were used as in A. (Gray) No changes; (dark blue) nucleoplasm higher; (light blue) cytoplasm higher. FDR < 5%, Fisher's exact test. At a lower cutoff (e.g., ≥1.5-fold), again, the same conclusion held that most m 6 A peaks were modified at the same level in nucleoplasmic mRNA and cytoplasmic mRNA.](https://www.researchgate.net/profile/Jacob-Hanna/publication/317783262/figure/fig5/AS:668594732036098@1536416814514/Each-individual-m-6-A-modification-is-modified-mostly-with-the-same-level-for-each-of-the_Q320.jpg)
![Figure 4. The majority of m 6 As is not located close to splice sites. (A) The density of m 6 A at increasing distances from 3 ′ or 5 ′ splice sites in CA-RNA (orange lines), the nucleoplasm (dark blue), and the cytoplasm (light blue). " Relative m 6 A peak density " for a fixed position from the splice site was calculated as the scaled m 6 A peak density at that position scaled proportional to the average m 6 A peak density in exonic regions >100 nt away from splice sites (black line). To clearly show the distribution of m 6 A peaks from the splice sites, we focused on internal exons with exon length at least 200 nt so that the 100-nt exon regions from the 5 ′ splice site ( " SS " ) and the 3 ′ splice site do not overlap. The internal exons 200-nt long contain ∼80% of all internal exon m 6 As. The center exon is required to have m 6 A. Exon number = 3069. Error bar is the SEM. (B) Seven percent of exonic m 6 As are within 50 nt of splice sites for internal exons in A. (Top left panel) Few m 6 As locate close to splice sites. (Top right panel) More RRACUs locate close to splice sites. Total RRACU number is 5673 in the same exons. P < 1 × 10 −24 , Fisher's exact test. (Bottom left panel) Long internal exons (≥200 nt, the internal exons in A) have 80% of total internal exon m 6 As, while short ones (<200 nt) have only the remaining 20%. (Bottom right panel) Long internal exons have only 28% of the total internal exon RRACU motifs (total RRACU number is 46,492 for total internal exons; P < 1 × 10 −100 , Fisher's exact test), in contrast to short ones that have the majority (72%). Even if we consider all m 6 A-containing internal exons (both long and short), still, only 20% of total internal exonic m 6 As are within 50 nt of splice sites. (C ) The CA-RNA m 6 A peaks that are higher showed higher density in exonic regions near 5 ′ or 3 ′ splice sites. " CA-RNA higher " refers to the m 6 A peaks in Figure 2A with higher m 6 A signal strength in CA-RNA (orange). " No change " refers to the frequency of m 6 A peaks with no difference between CA-RNAs and nucleoplasm RNAs in Figure 2A (gray). Error bars are SEM. (D) The majority (>90%) of transcripts in which m 6 As are higher in CA-RNA than nucleoplasmic RNA is at least 50 nt away from splice sites. A minority of CA-RNA transcripts does have a percentage greater density of m 6 A near splice sites (≤50 nt; 9% vs. 6%), but the majority of these modifications does not persist in nuceloplasmic RNA (Fig. 2A). (E) Internal constitutive exons containing m 6 A show no change in splicing between wild-type and Mettl3 knockout mouse ESCs. Internal constitutive exons with m 6 A are defined as the triexon structure, with constitutive exon being the center exon; at least one of the three exons should have m 6 A. All 8601 m 6 A-containing internal constitutive exons showed the same degree of exon inclusion in Mettl3 knockout and wild-type cells (significant changes are defined as ΔPSI [percent spiced in] ≥0.1; FDR <5%) despite a decrease in m 6 A level in mRNAs to 10% of wild type (Fig. 6A; Supplemental Fig. 11). (F ) A minority of internal alternative cassette exons with m 6 A (defined as the triexon structure, with alternative cassette exon being the center exon; at least one of the three exons should have m 6 A) shows splicing changes in Mettl3 knockout versus wild-type cells. Approximately 5% of all 1830 m 6 A-containing internal alternative cassette exons changed splicing in Mettl3 knockout cells (significant changes are defined as ΔPSI ≥0.1; FDR <5%). We also examined the splicing for all constitutive exons and alternative exons regardless of whether they contain m 6 A or not (i.e., even considering the indirect effects of m 6 A on splicing). Again, all 67,706 constitutive exons spliced identically, and only a very minor proportion (∼3%) of all 11,715 alternative cassette exons changed splicing. Other alternative splicing types showed even fewer changes, including alternative 5 ′ splice site, alternative 3 ′ splice site, and intron retention. We also analyzed the raw RNA-seq data of previous publications that reported certain splicing changes upon comprising Mettl3 expression levels (Supplemental Fig. 9; Dominissini et al. 2012; Zhao et al. 2014; Geula et al. 2015; Liu et al. 2015) and found the same result: that exons splice mostly the same when their exonic m 6 As were lost.](https://www.researchgate.net/profile/Jacob-Hanna/publication/317783262/figure/fig2/AS:508457339940864@1498237084924/The-majority-of-m-6-As-is-not-located-close-to-splice-sites-A-The-density-of-m-6-A-at_Q320.jpg)
