Systemic mRNA transport depends on m5C methylation, nuclear mRNA export factors and developmental phase changes

In Arabidopis a high number of distinct mRNAs move from shoot to root. We previously reported on the correlation of m5C-methylation and lack of mRNA transport in juvenile plants depending on the RNA methyltransferases DNMT2 NSUN2B. However, to our surprise we uncovered that lack of DNMT2 NSUN2B (writer) activity did not abolished transport of TCTP1 and HSC70.1 transcripts in flowering plants. We uncovered that transport of both transcripts is reinstated in dnmt2 nsun2b mutants after commitment to flowering. This finding suggests that additional factors are seemingly involved in regulating / mediating mRNA transport. In search of such candidates, we identified the two ALY2 and ALY4 nuclear mRNA export factors belonging to the ALYREF family as bona fide m5C readers mediating mRNA transport. We show that both proteins are allocated along the phloem and that they bind preferentially to mobile mRNAs. MST measurements indicate that ALY2 and ALY4 bind to mobile mRNAs with relative high affinity with ALY4 showing higher affinity towards m5C-methylated mobile mRNAs. An analysis of the graft-mobile transcriptome of juvenile heterografted-grafted wild type, dnmt2 nsun2b, aly2 and aly4 mutants revealed that the nuclear export factors are key regulators of mRNA transport. We suggest that depending on the developmental stage m5C methylation has a negative and positive regulatory function in mRNA transport and acts together with ALY2 and ALY4 to facilitate mRNA transport in both juvenile and flowering plants.