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Small RNA-seq from FPLC-fractionated leaf and flower crude extracts

ABSTRACT: Small RNA-seq from FPLC-fractionated leaf and flower crude extracts

PROVIDER: PRJNA540255 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8984470.fastq.gz	Fastqsanger.gz
	SRR8984480.fastq.gz	Fastqsanger.gz
	SRR8984471.fastq.gz	Fastqsanger.gz
	SRR8984481.fastq.gz	Fastqsanger.gz
	SRR8984472.fastq.gz	Fastqsanger.gz

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Small RNA-seq from FPLC-fractionated leaf and flower crude extracts

Project description:Background: RNA interference (RNAi) is an indispensable regulatory mechanism governing developmental processes and stress responses via sequence-specific control of target RNAs mediated by the action of small, 20–24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs. Biogenesis and sorting of miRNAs into ARGONAUTE (AGO) proteins are intensively investigated, however very few information is available about the presence and distribution of distinct sRNA pools in plant cells. Results: High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of sRNAs in the cytoplasm, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Investigation of some selected miRNAs in a transient expression system validated this finding. We also showed that the availability of RISCs is a limiting factor determining the loading efficiency of miRNAs. Conclusion: Our data reveal the existence of a regulatory checkpoint, likely controlled by information carried by the diverse miRNA precursors, determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.

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Project description:Wheat (Triticum ssp.) is one of the most important human food sources as well as livestock feed, but is very sensitive to changes in temperature. Specifically, processes during leaf growth and photosynthesis and during flower and seed development are affected by high temperature. While this has been investigated to some extent on physiological, developmental and molecular levels, very little is known about early signalling associated with an increase in temperature. Here, we probed the impact of an increase in temperature for 1 hour on the leaf and flower phosphoproteome of wheat. In order to identify differentially phosphorylated peptides, we used two wheat proteome databases, and could show the superiority of the new IWGSC genome. Our analyses of the leaf phosphoproteome further revealed differential phosphorylation of heat shock proteins, Photosystem I subunits, and plasma membrane intrinsic proteins, in addition to a number of potentially novel players.

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