Project description:Small RNAs recently emerged as a new class of mobile instructive signals in development. Here, we investigate their mechanism of action and show that the gradients formed by mobile small RNAs generate sharply defined domains of target gene expression. By modulating the source of artificial miRNAs we show that boundary formation is an inherent property of the small RNA gradient itself. The threshold-based readout of such gradients is highly sensitive to small RNA levels at the source, allowing plasticity in the positioning of a target gene expression boundary. In addition to generating sharp expression domains of their immediate targets, the readouts of opposing small RNA gradients enable formation of stable and uniformly positioned developmental boundaries. These novel patterning properties of small RNAs are reminiscent of those of morphogens in animal systems. However, their exceptionally high specificity, direct mode of action, and the fully intrinsic nature of their gradients, distinguish mobile small RNAs from classical morphogens. Our findings present mobile small RNAs and their targets as highly portable and evolutionarily-tractable regulatory modules through which to create pattern in development and beyond.
Project description:The Mobile CRISPRi system with and without mRFP-targeting sgRNA was engineered into Pseudomonas aeruginosa PA14 strain with chromosomally encoded mRFP. RNA was isolated from these strains, and the corresponding cDNA library was synthesized and sequenced in 150 bp paired-end reads. Approximately 1,000,000 reads were collected for each of the two samples, with ~94% alignment to PA14 WT by Bowtie254, and transcripts were counted with HTSeq55. Only genes with a non-normalized read count greater than 1 in both samples were included in analysis, with a coverage of 1286 genes (~20% genome). This data shows that the Mobile CRISPRi system is selective for sgRNA-guided knockdown of mRFP.
Project description:In RNA interference (RNAi), small-interfering (si)RNAs processed from double-stranded RNA guide ARGONAUTE(AGO) proteins to silence sequence-complementary RNA/DNA. Plant RNAi can propagate locally and systemically, but despite recent mechanistic advances, basic questions/hurdles remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata, yet how its dynamics in planta compares with that of established symplastic-diffusion markers remains unknown. Also unknown is why select siRNA species, or size-classes thereof, are recovered in RNAi-recipient tissues, yet only under some experimental settings. Finally, RNAi shootward movement in micro-grafted Arabidopsis necessary to study its presumptive transgenerational effects– has not been achieved thus far and endogenous functions of mobile RNAi remain scarcely documented. Focusing on non-amplified RNAi in Arabidopsis, we show here that (i) transgenic RNAi-movement, although symplasmic, only partially recapitulates the diffusion pattern of free GFP in planta, (ii) the presence/absence of specific AGOs in incipient/traversed/recipient tissues likely explains the apparent siRNA-selectivity observed during vascular movement, (iii) stress application allows endo-siRNA translocation against the shoot-to-root phloem flow, and (iv) mobile endo-siRNAs generated from a single inverted-repeat(IR) locus, have the potential to regulate hundreds of transcripts.