Identification of a cytoskeleton-associated 120 kDa RNA-binding protein in developing rice seeds

Area of Science:

• Plant Molecular Biology
• Seed Development Biology
• RNA Biology

Background:

• Prolamin RNAs localize to the surface of prolamine storage protein bodies (PBs) in rice seeds.
• The mechanism of prolamine RNA enrichment on the endoplasmic reticulum (ER) subdomain is not fully understood.
• Directed transport and targeting of RNAs to specific cellular locations involve RNA signal determinants and protein interactions.

Purpose of the Study:

• To investigate the molecular mechanisms underlying prolamine RNA localization during rice seed development.
• To identify proteins involved in the transport and targeting of prolamine RNAs to PBs.
• To characterize the function of a novel RNA-binding protein, Rp120.

Main Methods:

• Isolation of an enriched cytoskeleton-PB fraction from rice endosperm.
• RNA-binding UV-cross-linking assay to identify RNA-binding proteins.
• Recombinant cDNA cloning and sequence analysis of Rp120.
• Analysis of RNA-binding specificity using 5' UTR, 3' UTR, and coding sequences.
• Sucrose density gradient centrifugation to assess protein-protein interactions.

Main Results:

• A prominent RNA-binding activity, Rp120, was identified in the cytoskeleton-PB fraction.
• Rp120 shares structural homology with human p100 and possesses multiple nucleic acid-binding SN domains, a tudor domain, and a coiled-coil domain.
• Rp120 specifically binds the 3' UTR of prolamine RNA, but not its 5' UTR or coding sequences.
• Rp120 interacts with other proteins, suggesting an association with the cytoskeleton.

Conclusions:

• Rp120 is a novel RNA-binding protein implicated in the specific targeting of prolamine RNAs.
• The modular domain organization of Rp120 (SN, tudor, coiled-coil) supports its role in RNA processing and transport.
• Rp120 likely functions in RNA sorting within the rice endosperm, contributing to the proper localization of storage protein RNAs.