The cytoplasmic‐localized, cytoskeletal‐associated RNA binding protein OsTudor‐SN: evidence for an essential role in storage protein RNA transport and localization

Abstract

SummaryPrevious studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of OsTudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. OsTudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that OsTudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that OsTudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of OsTudor-SN particles in transgenic rice plants expressing GFPtagged prolamine RNA transport particles showed co-localization of OsTudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged OsTudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of OsTudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that OsTudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.

Translational Regulation of Cytoplasmic mRNAs

“…Glutelin mRNAs have their own zipcode elements (Hamada et al, 2003b;Washida et al, 2009). Both types of mRNA form cytoskeleton associated granules that move along cytoskeletal elements (Muench et al, 1998;Muench et al, 2000;Okita and Choi, 2002;Wang et al, 2008;Washida et al, 2009).…”

Multifunctional RNA Binding Protein OsTudor-SN in Storage Protein mRNA Transport and Localization

“…This RBP was observed as particles whose size and movement were reminiscent of the movement observed for GFP-tagged MS2-prolamine mRNA particles (Hamada et al, 2003). Indeed, double immunofluorescence studies showed that OsTudor-SN colocalized with GFP-MS2 mRNA particles (Wang et al, 2008). These lines of evidence suggest that OsTudor-SN participates in the transport and localization of storage protein mRNAs.…”

“…The in vivo association of OsTudor-SN to the storage protein mRNAs was confirmed by the analysis of RNAs associated with immunoprecipitates (IPs) generated by purified OsTudor-SN antibody (Wang et al, 2008). Further immunofluorescence microscopy studies showed that OsTudor-SN is distributed to the PB-ER and cisternal-ER, suggesting a role in the localization of storage protein mRNAs.…”

“…The three pathways, however, are interrelated, as mutations in the glutelin mRNA transport pathway redirect glutelin mRNAs from the cisternal-ER to the PB-ER (Crofts et al, 2005;Doroshenk et al, 2010), while a mutation in the prolamine mRNA transport pathway mislocalizes prolamine mRNAs from the PB-ER to the cisternal-ER (Crofts et al, 2005;Fukuda et al, 2016). In addition to zip code-specific trans-factors, other ancillary factors are required for mRNA transport and localization, as suggested for the rice OsTudorStaphylococcus nuclease (SN; Wang et al, 2008).…”

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay

“…TSN also interacts with small ribonucleoproteins and Sm proteins involved in pre-mRNA splicing Gao et al, 2012;Cappellari et al, 2014). In plants, TSN is an RNA-binding protein involved in RNA transport and localization, which is essential for stress tolerance and has been linked to the formation of stress granules and processing bodies (Wang et al, 2008;Frei dit Frey et al, 2010;Gutierrez-Beltran et al, 2015). Hence, TSN functions as a scaffold for protein-protein or protein-RNA interactions in transcription regulation, mRNA localization and pre-mRNA splicing (Gutierrez-Beltran et al, 2016).…”