Multiple RNA Binding Protein Complexes Interact with the Rice Prolamine RNA Cis-Localization Zipcode Sequences

Abstract

RNAs for the storage proteins, glutelins and prolamines, contain zipcode sequences, which target them to specific subdomains of the cortical endoplasmic reticulum in developing rice (Oryza sativa) seeds. Fifteen RNA binding proteins (RBPs) specifically bind to the prolamine zipcode sequences and are likely to play an important role in the transport and localization of this storage protein RNA. To understand the underlying basis for the binding of multiple protein species to the prolamine zipcode sequences, the relationship of five of these RBPs, RBP-A, RBP-I, RBP-J, RBP-K, and RBP-Q, were studied. These five RBPs, which belong to the heterogeneous nuclear ribonucleoprotein class, bind specifically to the 59 coding regions as well as to the 39 untranslated region zipcode RNAs but not to a control RNA sequence. Coimmunoprecipitation-immunoblot analyses in the presence or absence of ribonuclease showed that these five RBPs are assembled into three multiprotein complexes to form at least two zipcode RNA-protein assemblies. One cytoplasmic-localized zipcode assembly contained two multiprotein complexes sharing a common core consisting of RBP-J and RBP-K and either RBP-A (A-J-K) or RBP-I (I-J-K). A second zipcode assembly of possibly nuclear origin consists of a multiprotein complex containing RBP-Q and modified forms of the other protein complexes. These results suggest that prolamine RNA transport is initiated in the nucleus to form a zipcode-protein assembly, which is remodeled in the cytoplasm to target the RNA to its proper location on the cortical endoplasmic reticulum.

Comprehensive proteomic analysis of developing protein bodies in maize (Zea mays) endosperm provides novel insights into its biogenesis

“…This is perhaps not surprising because HnRNPs predominantly shuttle between the nucleus and the cytosol to participate in mRNA biogenesis and mRNA export from the nucleus (Yeap et al , 2014). Rice RBP-A, RBP-I, RBP-J, RBP-K, and RBP-Q were consistently distributed into both the nucleus and the cytoplasm (Yang et al , 2014). In particular, RBP-A co-localized with the nucleus, the cisternal ER membrane, and microtubules but not with the PB-ER (Crofts et al , 2010).…”

“…This suggests that these HnRNPs might primarily function in export of prolamine RNAs from the nucleus and transport in the cytosol before their localization in the PB-ER. Moreover, they could form several protein complexes (A–J–K, I–J–K) on the prolamine zipcode from the nucleus to the cytoplasm (Yang et al , 2014). It should be pointed out that two members (RBP-A and K) of the complexes have no corresponding orthologs in the maize genome.…”

mRNA Localization in Plant Cells

“…Using the prolamine zipcode sequences as bait, 15 RBPs were selectively captured under stringent binding-elution conditions (Crofts et al, 2010). Followup functional studies of five of these captured proteins, RBPs A, I, J, K, and Q, exhibited direct binding to prolamine zipcode RNA and assembled into at least three zipcode RNA-protein complexes (Yang et al, 2014). RBPs A-J-K and I-J-K formed two complexes, while Q formed a third complex with other unidentified proteins (Yang et al, 2014).…”

“…Followup functional studies of five of these captured proteins, RBPs A, I, J, K, and Q, exhibited direct binding to prolamine zipcode RNA and assembled into at least three zipcode RNA-protein complexes (Yang et al, 2014). RBPs A-J-K and I-J-K formed two complexes, while Q formed a third complex with other unidentified proteins (Yang et al, 2014). The finding of multiple complexes is consistent with the dynamic remodeling of mRNP complexes in the multistep process of mRNA localization.…”

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

“…Further details on BiFC can be found in Supplemental Table S1. Tobacco BY2 cells were used for transient expression to examine the protein-protein interaction based upon the generation of EYFP fluorescence using methods described previously (Yang et al, 2014).…”