Chemical modification of transcripts with 5′ caps occurs in all organisms. Here, we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of an organism's cap epitranscriptome. The method was piloted with 21 canonical caps—m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG—and 5 ‘metabolite’ caps—NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus, Escherichia coli, yeast, mouse tissues, and human cells, we discovered new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and high proportions of caps lacking 2′-O-methylation (m7Gpppm6A in mammals, m7GpppA in dengue virus). While substantial Dimroth-induced loss of m1A and m1Am arose with specific RNA processing conditions, human lymphoblast cells showed no detectable m1A or m1Am in caps. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps.

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6 7 Chemical modification of transcripts with 5' caps occurs in all organisms. Here we report a 8 systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of the 9 cap epitranscriptome in any organism. The method was piloted with 21 canonical caps -10 m 7 GpppN, m 7 GpppNm, GpppN, GpppNm, and m 2,2,7 GpppG -and 5 "metabolite" caps -NAD, 11 FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue 12 virus, Escherichia coli, yeast, mice, and humans, we discovered four new cap structures in 13 humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m 7 Gpppm 6 A), cell-and tissue-specific 14 variations in cap methylation, and surprisingly high proportions of caps lacking 2'-O-methylation, 15 such as m 7 Gpppm 6 A in mammals and m 7 GpppA in dengue virus, and we did not detect cap 16 m 1 A/m 1 Am in humans. CapQuant accurately captured the preference for purine nucleotides at 17 eukaryotic transcription start sites and the correlation between metabolite levels and metabolite 18 caps. The mystery around cap m 1 A/m 1 Am analysis remains unresolved. 19 20 21 cap structure involves -phosphate methylation of unprocessed 5'-triphosphate (mPPPN) on 1 small RNAs such as mammalian U6 and 7SK, mouse B2, and plant U3 RNAs (7). 2 A variety of non-canonical caps involving nucleotide metabolites (Figure 1A) have also recently 3 been described (8,9). For example, nicotinamide adenine dinucleotide (NAD) and coenzyme A 4 (CoA) were found as cap-like structures in bacterial small RNAs (10) and the NAD cap was also 5 found in yeast and human mRNA and non-coding RNAs (11). Julius and Yuzenkova expanded 6 the potential repertoire of caps by demonstrating that a variety of nucleotide metabolites could 7 initiate transcription by bacterial RNA polymerase (RNA Pol) in vitro, including flavin adenine 8 dinucleotide (FAD), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate N-9 acetylglucosamine (UDP-GlcNAc) (9). They also showed that capping with NAD and UDP 10 analogs by bacterial RNA Pol is promoter-specific and stimulates promoter escape (9), 11 suggesting a role for metabolite caps in regulating gene expression. For example, the NAD cap 12 has been shown to influence RNA stability and turnover, and is a substrate for decapping 13 enzymes (11). However, the lack of sensitive and specific analytical methods has hindered the 14 systematic study of the cap landscape dynamics in cells. 15Analysis of RNA cap structures has traditionally relied on radioisotope labeling and enzymatic 16 hydrolysis, followed by thin-layer and other types of chromatography to resolve cap structures 17 (12)(13)(14). While sensitive, the radiolabeling approach lacks specificity (12) and has the potential 18 to create cellular toxicity artifacts (15,16). While two-dimensional electrophoresis (14) allows 19 multiple caps analysis, it (i) lacks specificity for identifying intact cap structures, (ii) is limited to 20 NpppN caps, (iii) does not provide absolute quantification, and (iv) is semi-quantitative at b...

Intercropping, a traditional and worldwide cropping practice, has been considered as a paradigm of sustainable agriculture based on complementary mechanisms among different crop species. Soybean (Glycine max) is widely relay intercropped about 60 days before maize (Zea mays) harvest in Southwest China. However, shade caused by tall maize plants may be a limiting factor for soybean growth at a seedling stage. In field research, we studied the ecophysiological responses of two widely cultivated soybean varieties [Gongqiudou494-1 (GQD) and Gongxuan 1 (GX)] to maize shading in a relay intercropping system (RI) compared with monocropped soybean plants (M). Our results showed that soybean seedlings intercropped with maize exhibited significantly downregulated net photosynthetic rate (P N ) (-38.3%), transpiration rate (-42.7%), and stomatal conductance (-55.4%) due to low available light. The insignificant changes in intercellular CO 2 concentration and the maximum efficiency of PSII photochemistry suggested that the maize shading-induced depressions in P N were probably caused by the deficiency of energy for carbon assimilation. The significantly increased total chlorophyll (Chl) content (+27.4%) and Chl b content (+52.2%), with lowered Chl a/b ratios (-20.5%) indicated soybean plants adjusted their light-harvesting efficiency under maize shading condition. Biomass and leaf area index (LAI) of seedlings under RI decreased significantly (-78.7 and -71%, respectively) in comparison with M. Correlation analysis indicated the relative reduction in biomass accumulation was caused by the decline in LAI rather than P N , it affected negatively the final yields of soybean (32.8%). Cultivar-specific responses to maize shading were observed in respects of LAI, biomass, and grain yield. It indicated that GX might be a better cultivar for relay intercropping with maize in Southwest China.

Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used CRISPR/Cas9-mediated high-throughput genetic screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Gué rin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens can elucidate highly complex hostpathogen interactions and serve to identify HDTs with the potential to improve TB treatment.