Regulatory RNAs (MLU-associated group)

We are using several in silico, in vitro and in planta approaches to define the role of lncNATs in the context of multigene families of A. thaliana and to identify the processes in which they are involved. These approaches include the analysis of available mutants and the generation of reporter, overexpression, knock-down and knock-out lines to figure out the function of the lncNATs and to characterize the generated transcripts and their potential target(s) using a combination of molecular biology, biochemistry, transcriptomics, proteomics and bioinformatics. Our studies of two lncNATs overlapping genes of the uridine diphosphate glycosyltransferases (UGTs) superfamily and one overlapping a member of the AUX/IAA proteins family provide insights about the action of lncNATs in a multigene family context.

According to our initial hypothesis, transient expression assays in tobacco leaves showed that co-expression of lncNATs leads to production of small RNAs and to downregulation of primary and closely related secondary targets. Surprisingly, the results are different in the host plant A. thaliana, in which lncNATs overexpression or downregulation does not affect the expression levels of the same targets. These results suggest that, at least in these cases, silencing is not occurring in the original host, a fact that could explain why sense and antisense genes can be co-expressed in particular tissues.

Alternatively, alteration of lncNATs expression levels can trigger morphological changes. Our results indicate that overexpression and downregulation of two lncNATs that overlap one of the UGT genes results in plants with increased or decreased rosette area, respectively. Microscopic analysis showed that this phenotype is consequence of changes in cell size, an effect that is more pronounced at the bottom of the leaves, where the lncNATs are expressed. We confirmed that this phenotype is due to the RNA molecule acting as bona fide lncRNA and not a result of the action of small peptides encoded in its sequence.

For the two other lncNATs under study we identified that one of them acts in cis regulating the expression of the sense gene, which is involved in salt-stress responses and defense against the fungal pathogen Botrytis cinerea, and have preliminary evidences that the second lncNAT acts in trans, modulating translation of the sense mRNA.

Several lncRNAs achieve their function via interaction with proteins and their identification is central to infer in which biological processes are the lncRNAs involved. In order to identify proteins that interact with lncNATs, we developed an RNA-centric method that allows the capture of proteins using as baits lncNATs synthetized in vitro. Additionally, and taking advantage of several previously generated transgenic lines, we established an in vivo approach in which the interactions that occur in the plant cell are fixed via crosslinking, avoiding unspecific interactions that are prone to be produced in vitro. The identification of the interacting proteins is performed by mass-spectrometry.

With the aim to generate plants with increased resistance to pathogens, we actively collaborate with Prof. Behrens, from the Martin Luther University, in the development of the project “A new technique to protect plants against virus infections”. This project is focused in the design, optimization and use of RNA-based tools for the control of viral infections. By using in silico and in vitro approaches, we identified virus-derived small RNAs efficient for protection and confirmed their activity in planta. Based on the success of the mentioned strategy, we aim to extend its use for the control of other plant pathogens.

Reference:
Gago-Zachert S, Schuck J, Weinholdt C, Knoblich M, Pantaleo V, Grosse I, Gursinsky T, Behrens SE. Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro. Nucleic Acids Res. 2019 Sep 26;47(17):9343-9357. doi: 10.1093/nar/gkz678.