Enod40, a Short Open Reading Frame–Containing mRNA, Induces Cytoplasmic Localization of a Nuclear RNA Binding Protein in Medicago truncatula

Abstract

In eukaryotes, diverse mRNAs containing only short open reading frames (sORF-mRNAs) are induced at specific stages of development. Their mechanisms of action may involve the RNA itself and/or sORF-encoded oligopeptides. Enod40 genes code for highly structured plant sORF-mRNAs involved in root nodule organogenesis. A novel RNA binding protein interacting with the enod40 RNA, MtRBP1 (for Medicago truncatula RNA Binding Protein 1), was identified using a yeast three-hybrid screening. Immunolocalization studies and use of a MtRBP1-DsRed2 fluorescent protein fusion showed that MtRBP1 localized to nuclear speckles in plant cells but was exported into the cytoplasm during nodule development in enod40-expressing cells. Direct involvement of the enod40 RNA in MtRBP1 relocalization into cytoplasmic granules was shown using a transient expression assay. Using a (green fluorescent protein)/MS2 bacteriophage system to tag the enod40 RNA, we detected in vivo colocalization of the enod40 RNA and MtRBP1 in these granules. This in vivo approach to monitor RNA-protein interactions allowed us to demonstrate that cytoplasmic relocalization of nuclear proteins is an RNA-mediated cellular function of a sORF-mRNA.

A stable tRNA-like molecule is generated from the long noncoding RNA GUT15 in Arabidopsis

“…In the same study, additional 26 small ORFs were identified in annotated lncRNAs. These are not the only cases of transcripts believed to solely function as RNA molecules that do in fact code for small peptides [12,36]. In our studies we also detected the AtGUT15 transcripts to be attached to translating ribosomes.…”

“…In rice, the LDMAR noncoding transcript is required for normal male fertility under long-day conditions [11]. Plant lncRNAs are also involved in root nodule organogenesis; for example, the ENOD40 lncRNA plays a role in the cytoplasmic re-location of the nuclear protein RBP1 during nodulation in Medicago truncatula [12]. Bardou and coworkers [13] identified that the ASCO lncRNA is a novel player (competitor) in the regulation of alternative splicing.…”

The Requirement of c-Jun N-terminal Kinase 2 in Regulation of Hypoxia-inducing Factor-1α mRNA Stability

“…The biotinylated RNAs were incubated with cell lysis from WT MEFs (in PLB buffer) in a final volume of 200 l at room temperature for 30 min and captured by streptavidin MagneSphere Paramagnetic Particle (Promega). After washing according to the manufacturer's instructions, bound proteins were eluted in Laemmli buffer and resolved by SDS-PAGE, electrically transferred to PVDF membrane, and the presence of nucleolin and HuR was detected with Western blotting assay (44).…”

Non-protein coding RNAs, a diverse class of gene regulators, and their action in plants

“…This npcRNA seems to be required for correct re-localization of MtRBP1, a constitutively expressed RNA-binding protein, from the nucleus to cytoplasmic granules, acting as a molecular cargo like the meiRNA does in yeast. 68 Many new npcRNAs are being identified in plants. For example, in our laboratory, we have used a genome-wide bioinformatic analysis to screen full-length cDNA databases in Arabidopsis.…”