The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation.

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3, and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady-state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2.

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.

Genes are strings of genetic code that contain instructions for producing a cell's proteins. Active genes are copied from DNA into molecules called mRNAs, and mRNA molecules are subsequently translated to create new proteins. However, the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA. Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production. One of these methods involves adding a chemical tag, a methyl group, onto mRNA while it is being created. These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation. Most eukaryotic species ­ which include animals, plants and fungi ­ use the same system to add methyl tags to mRNA molecules. One methyl tag in particular, known as m⁶A, is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants. However, in the flowering plant Arabidopsis thaliana, ECT2 was thought to bind to an mRNA sequence different from the one normally carrying the chemical tag, creating obvious confusion about how the system works in plants. Arribas-Hernández, Rennie et al. investigated this question using advanced large-scale biochemical techniques, and discovered that conventional m⁶A methyl tags are indeed used by ECT2 in Arabidopsis thaliana. The confusion likely arose because the sequence ECT2 was thought bind is often located in close proximity to the m⁶A tags, possibly acting as docking stations for proteins that can influence the ability of ECT2 to bind mRNA. Arribas-Hernández, Rennie et al. also uncovered additional mRNA sequences that directly interact with parts of ECT2 previously unknown to participate in mRNA binding. These findings provide new insights into how chemical labels in mRNA control gene activity. They have broad implications that extend beyond plants into other eukaryotic species, including humans. Since this chemical labelling system has a major role in controlling plant growth, these findings could be leveraged in biotechnology applications to improve crop yields and enhance plant-based food production.

Transport of Live Cells Under Sterile Conditions Using a Chemotactic Droplet.

1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated if such droplets can be used as transporters for living cells. We developed a partially hydrophobic alginate capsule as a protective unit that can be precisely placed in a droplet and transported along chemical gradients. Once the droplets with cargo reached a defined final destination, the association of the alginate capsule and decanol droplet was disrupted and cargo deposited. Both Escherichia coli and Bacillus subtilis cells survived and proliferated after transport even though transport occurred under harsh and sterile conditions.