Evolution of the Small Family of Alternative Splicing Modulators Nuclear Speckle RNA-Binding Proteins in Plants
, MartĂn D. Crespi 2,* and Federico Ariel 1,*Round 1
Reviewer 1 Report
Dear authors,
It was nice to read about the NSRs and the ofcus you haven given your study. Although we can always do more, I think the story you told sounds already interesting. Therefore i have no further comments for the publications so far.
Author Response
We thank the Reviewer for his/her positive comment on our work.
Reviewer 2 Report
Nuclear Speckle RNA-binding proteins (NSRs) were first identified as protein partners of the symbiotic nodulation ENOD40 lncRNA and subsequently shown in Arabidopsis to play a role as alternative splicing modulators of specific mRNAs with a role in auxin driven lateral root formation. NSRs are localized in nuclear speckles where components of the splicing machinery accumulate in plant cells but display a cytosolic granule distribution on interaction with ENOD40. They also recognize the lncRNA ASCO (ALTERNATIVE SPLICING COMPETITOR) that regulates lateral root formation, where ASCO was found to compete for NSRs interaction with some of their alternatively spliced mRNA targets. In this work, authors explored the phylogenetic history of NSRs and show that these RNA-binding proteins acquired particular domains throughout evolution, and have undergone in Eudicots an evolutionary reductive trend which may be compensated by alternative splicing variants.
NSRs of Eudicots share an NLS motif, a conserved motif of unknown function and the C-terminal RNA Recognition (RRM) domain. RNA recognition is associated to two motifs: motif 1 present in all NSRs with exception of Selaginella; and motif 2 that is present in Monocots and Eudicots but not in Amborella. Notably, Selaginella NSRs display a related motif 9 that is also present in Marchantia, which indicates that a partial RRM predates the origin of land plants, and was completed in flowering plants by motif 2, before the split of Monocots and Dicots. Motifs 3 and 6, corresponding to the conserved motif of unknown function and the NLS, are on the other hand exclusive of Angiosperms. Thus, authors propose that these proteins may have acquired a nuclear localization in Angiosperms, whereas NSRs of Marchantia and Selaginella are most likely cytoplasmic and eventually do not participate in alternative splicing.
Expression studies of the two Medicago and L. japonicus NSR genes during rhizobial nodulation showed that even their transcript levels remain constant during nodulation, NSR1 is more abundant in the nitrogen fixation zone, while NSR2 is higher in the meristematic zone, in opposite to ENOD40 which accumulates throughout the whole nodule. By comparing the RNA populations of nodules and non-inoculated roots, authors actually identify an important number of alternative 3’-ends and intron retention events. Remarkably, frequency of these events does change across samples, but a progression in the number of affected genes is observed between mature nodules and nodule primordia, coinciding with NSRs and ENOD40-2 downregulation and the induction of ENOD40-1. Based on these findings and the differential expression of NSR1/NSR2 genes throughout nodulation, authors propose a prevalent role of the NSR-ENOD40 machinery modulating alternative splicing in legume symbiosis, by fine-tuning the accumulation of specific mRNA isoforms governing the different stages of nodule organogenesis.
Overall, these are very relevant results that hint a prevalent role of members of the NSR family and their lncRNA partners in modulating alternative mRNA splicing in different developmental contexts and in promoting accumulation of specific protein isoforms. The manuscript is well written and studies carefully performed and as such the work merits acceptance for publication. Concerning role of Marchantia and Selaginella NSRs, it may be relevant including transitory studies in N. benthamiana to analyze whether these proteins display or not a nuclear localization, and if they are able to form nuclear speckles as Eudicot NSRs. Further description of the genes observed to undergo alternative splicing during progression of nodule development would also reinforce the role of NSRs in legume symbiosis and in the regulation of genes governing nodule development.
Author Response
We thank the Reviewer for his/her positive feedback about our work.
Two main suggestions were made by the Reviewer:
Concerning role of Marchantia and Selaginella NSRs, it may be relevant including transitory studies in N. benthamiana to analyze whether these proteins display or not a nuclear localization, and if they are able to form nuclear speckles as Eudicot NSRs.
R/ This is a very constructive suggestion. However, such experiments would require cloning from different plant species before performing the sub-cellular localization assays, what is out of the scope of our work. We have now included in the discussion a phrase about the future perspectives related to the lack of the NLS in certain NSR proteins.
Further description of the genes observed to undergo alternative splicing during progression of nodule development would also reinforce the role of NSRs in legume symbiosis and in the regulation of genes governing nodule development.
R/ We agree about the relevance of this point. We have now included a detailed Table S3 with all the AS events identified in Medicago and Lotus for the sake of the scientific community.
