Burgardt, R. | Search bioRxiv

bioRxiv2026

A structured RNA balances DEAD-box RNA helicase function in plant alternative splicing control

Eukaryotic gene expression is a multi-layered process influenced by multiple factors. One of them is the secondary structure of precursor mRNAs that can impact various aspects of their processing including alternative splicing (AS). Here, we report the functional characterization of the conserved RNA structural element DEAD that is located in DEAD-box RNA helicase (DRH) genes from land plants and serves as a sensor for RNA helicase activity by controlling AS. In Arabidopsis thaliana, it is found in DRH1 and its closest paralog, regulating usage of an alternative splice site as part of a negative feedback loop. Accordingly, opening of the structure shifts splicing towards non-coding variants, thereby balancing transcript and protein levels. Interestingly, the system is specific to DRH1 and its paralog and does not react to related helicases, which is at least partially conferred by the disordered and RGG/RG motif-containing C-terminus of DRH1. The importance of DEAD is underlined by the observation that releasing this attenuation mechanism causes massive changes in AS - mainly intron retention and exon skipping - and gene expression and results in a severe stress phenotype. Thus, DEAD provides a critical buffering mechanism to fine-tune helicase levels and their global impact on RNA structure-responsive gene expression.

plant biology↗

bioRxiv2025

The structured mRNA element 45ABC mediates auto- and cross-regulation of RBP45 genes via alternative splicing

Alternative splicing (AS) is a common gene regulatory mechanism involving distinct interactions between trans-acting factors and cis-regulatory elements on the precursor mRNA (pre-mRNA). In this study, we have functionally characterized the structured motif 45ABC, which is located in the pre-mRNAs of RNA-binding protein (RBP) 45 genes in many plant species. Our data revealed that this element mediates a negative auto- and cross-regulatory feedback loop via AS of the three 45ABC-containing RBP45 genes in Arabidopsis thaliana. We identified a G-rich stretch within the first stem as a potential RBP45 binding site and observed increased RBP45-dependent AS upon structural weakening of this pairing element. The second stem includes the alternative 5 splice site being activated in the presence of RBP45. Based on the known interaction between RBP45 homologs and U1 snRNP components required for 5 splice site recognition, we propose that RBP45 binding to stem I of 45ABC induces usage of the alternative 5 splice site in stem II. The resulting splicing variant is unproductive, thereby diminishing RBP45 expression. Analysing the splicing regulatory impact of the three At-RBP45 genes in auto- and cross-regulation and a transcriptome-wide manner revealed unequal genetic redundance with a major role of RBP45B. Furthermore, phenotypical analysis of single and higher order rbp45 mutants pointed at these genes functions in controlling primary root growth and flowering time. Taken together, we demonstrated that both sequence and structural features of 45ABC are critical for proper splicing control, balancing RBP45 expression and functions in plants via a conserved mRNA motif. Significance statementFunctional characterisation of a structured mRNA motif present in plant RBP45 genes identified sequence and pairing elements underlying a negative auto- and cross-regulatory expression circuit on the level of alternative splicing. Our study provides a rationale for the evolutionary conservation of this RNA element, which allows balancing levels and functions of RBP45 proteins as a requirement for proper plant development.

plant biology↗