Proteomic Analysis of Cytoskeleton-Associated RNA Binding Proteins in Developing Rice Seed

Doroshenk, Kelly A.; Crofts, Andrew J.; Morris, Robert T.; Wyrick, John J.; Okita, Thomas W.

doi:10.1021/pr900537p

Cited by 34 publications

(40 citation statements)

References 122 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Performing capture experiments in the absence of heparin led to the detection of many more proteins (data not shown), including the RNA binding protein Tudor-SN (Rp120) which is known to be eYciently captured on a heparin column (Sami-Subbu et al 2001). Such reduced stringency capture experiments have recently been used to more generally assess the role of cytosolic, cytoskeleton-associated RNA binding proteins (Doroshenk et al 2009). …”

Section: Discussionmentioning

confidence: 99%

Isolation and identification of cytoskeleton-associated prolamine mRNA binding proteins from developing rice seeds

Crofts

Whitelegge³

et al. 2010

Planta

Self Cite

View full text Add to dashboard Cite

The messenger RNA of the rice seed storage protein prolamine is targeted to the endoplasmic reticulum (ER) membranes surrounding prolamine protein bodies via a mechanism, which is dependent upon both RNA sorting signals and the actin cytoskeleton. In this study we have used an RNA bait corresponding to the previously characterized 5'CDS prolamine cis-localization sequence for the capture of RNA binding proteins (RBPs) from cytoskeleton-enriched fractions of developing rice seed. In comparison to a control RNA, the cis-localization RNA bait sequence led to the capture of a much larger number of proteins, 18 of which have been identified by tandem mass spectrometry. Western blots demonstrate that several of the candidate proteins analyzed to date show good to excellent specificity for binding to cis-localization sequences over the control RNA bait. Temporal expression studies showed that steady state protein levels for one RNA binding protein, RBP-A, paralleled prolamine gene expression. Immunoprecipitation studies showed that RBP-A is bound to prolamine and glutelin RNAs in vivo, supporting a direct role in storage protein gene expression. Using confocal immunofluorescence microscopy, RBP-A was found to be distributed to multiple compartments in the cell. In addition to the nucleus, RBP-A co-localizes with microtubules and is associated with cortical ER membranes. Collectively, these results indicate that employing a combination of in vitro binding and in vivo binding and localization studies is a valid strategy for the identification of putative prolamine mRNA binding proteins, such as RBP-A, which play a role in controlling expression of storage protein mRNAs in the cytoplasm.

show abstract

Section: Discussionmentioning

confidence: 99%

Isolation and identification of cytoskeleton-associated prolamine mRNA binding proteins from developing rice seeds

Crofts

Whitelegge³

et al. 2010

Planta

Self Cite

View full text Add to dashboard Cite

show abstract

“…Samples were focused on an Ettan IPGphor 3 System (GE Healthcare) with a ceramic manifold as follows: 500 V for 66 min; gradient to lOOOV for 66 min; gradient to lOkV for 3 hrs, 18 min; 10 kV until 50000 total Vhrs were reached. After IEF, strips were equilibrated and mn on 10-18% polyacrylamide gradient gels as in Doroshenk et al (2009). For preparative 2D gels, 1 mg of protein was diluted to 425 \x\ with sample buffer and 1% total carrier ampholytes and run as described above.…”

Section: Protein Sample Preparation Cydye Labeling and 2d Gel Eleetmentioning

confidence: 99%

“…Corresponding protein spots from preparative Sypro Ruby-stained (Bio-Rad) 2D gels were excised and in-gel trypsin digested as in Crofts et al (2010). Extraeted peptides were analyzed by liquid chromatographytandem mass spectrometry as described in Doroshenk et al (2009).…”

Section: D Gel Image Analysis and Protein Identificationmentioning

confidence: 99%

Characterization of the rice glup4 mutant suggests a role for the small GTPase Rab5 in the biosynthesis of carbon and nitrogen storage reserves in developing endosperm

et al. 2010

Self Cite

View full text Add to dashboard Cite

Rice is unique among plants in that it accumulates major amounts of two types of seed storage proteins, prolamines and glutelins, which along with starch, serve as sources of carbon and nitrogen for the postgerminative seedling. Here, we investigate the role of the small GTPase Rab5 in the biosynthesis of storage proteins through characterization of the loss-of-function mutant glup4. In situ RT-PCR and fiuorescence microscopy revealed the glup4 mutant is impaired in glutelin mRNA localization to the cortical endoplasmic reticulum in developing endosperm cells. Furthermore, microarray and two-dimensional difference in gel electrophoresis analysis of transcript and protein abundance, respectively, identified a number of genes whose expression is affected by the loss of Rab5, including starch biosynthetic enzymes. Our results indicate Rab5 is important for RNA and protein sorting in rice seed and supports previous observations that the biosynthesis of starch and storage proteins may be interrelated.

show abstract

“…Expansin, a cell wall protein associated with cellular expansion and cell wall changes, and a transcription elongation factor (TFIIS) that stimulates RNA polymerase II also were identified. By contrast, the Tsn peptide module interacted with eight RBPs, RBP-23, RBP-43, RBP-59, RBP-61, RBP-100, RBP-152, RBP-157, and RBP-159 (Table I), identified in earlier studies from this laboratory (Doroshenk et al, 2009(Doroshenk et al, , 2012 as major RBPs present in developing rice endosperm. These findings suggest that the two OsTudor-SN modules play distinct biological roles.…”

Section: Resultsmentioning

confidence: 82%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

The multifunctional RNA-binding protein Tudor-SN plays multiple roles in transcriptional and posttranscriptional processes due to its modular domain structure, consisting of four tandem Staphylococcus nuclease (SN)-like domains (4SN), followed by a carboxyl-terminal Tudor domain, followed by a fifth partial SN sequence (Tsn). In plants, it confers stress tolerance, is a component of stress granules and P-bodies, and may participate in stabilizing and localizing RNAs to specific subdomains of the cortical-endoplasmic reticulum in developing rice (Oryza sativa) endosperm. Here, we show that, in addition to the intact rice OsTudor-SN protein, the 4SN and Tsn modules exist as independent polypeptides, which collectively may coassemble to form a complex population of homodimer and heteroduplex species. The 4SN and Tsn modules exhibit different roles in RNA binding and as a protein scaffold for stress-associated proteins and RNA-binding proteins. Despite their distinct individual properties, mutations in both the 4SN and Tsn modules mislocalize storage protein mRNAs to the cortical endoplasmic reticulum. These results indicate that the two modular peptide regions of OsTudor-SN confer different cellular properties but cooperate in mRNA localization, a process linking its multiple functions in the nucleus and cytoplasm.mRNA localization is readily evident in structurally polarized somatic cells

show abstract

Proteomic Analysis of Cytoskeleton-Associated RNA Binding Proteins in Developing Rice Seed

Cited by 34 publications

References 122 publications

Isolation and identification of cytoskeleton-associated prolamine mRNA binding proteins from developing rice seeds

Isolation and identification of cytoskeleton-associated prolamine mRNA binding proteins from developing rice seeds

Characterization of the rice glup4 mutant suggests a role for the small GTPase Rab5 in the biosynthesis of carbon and nitrogen storage reserves in developing endosperm

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

Contact Info

Product

Resources

About