Detection and in vitro studies of Cucurbita maxima phloem serpin-1 RNA-binding properties.

Apart from being a conduit for photoassimilate transport in plants, the phloem serves as a pathway for transport of proteins and RNAs from sites of their synthesis to distant plant parts. As demonstrated for mRNAs and small RNAs such as miRNA and siRNA, their phloem transport is largely involved in responses to environmental cues including stresses and pathogen attacks. RNA molecules are believed to be transported in the phloem in the form of complexes with RNA-binding proteins; however, proteins forming such complexes are generally poorly studied. Here, we demonstrate that the Cucurbita maxima phloem serpin-1 (CmPS1), which has been previously described as a functional protease inhibitor capable of long-distance transport via the phloem, is able to bind RNA in vitro. Among different RNAs tested, CmPS1 exhibits a preference for imperfect RNA duplexes and the highest affinity to tRNA. A characteristic complex formed by CmPS1 with tRNA is not observed upon CmPS1 binding to tRNA-like structures of plant viruses. Mutational analysis demonstrates that the CmPS1 N-terminal region is not involved in RNA binding. Since antithrombin-III, the human protease inhibitor of serpin family most closely sequence-related to CmPS1, is found to be unable to bind RNA, one can suggest that, in its evolution, CmPS1 has gained the RNA binding capability as an additional function likely relevant to its specific activities in the plant phloem.

Data on the exon-intron organization of genes coding for B-cell receptor-like proteins.

B-cell receptor-associated protein (BAP) family plays important roles in the ER homeostasis and stress responses of eukaryotic cells [1]. We reported the analysis of plant BAP-like (PBL) genes and the encoded proteins of higher land plants [2]. The origin and functional divergence of these genes among all eukaryotes, however, are poorly studied, which impedes our understanding of the functional relationships and diversity among BAP-like proteins. One possible reason for the potential functional diversity may be the differences in the exon-intron structure of PBL genes. In this study, we first performed analysis of the exon-intron organization of these genes in the genome sequences of the Viridiplantae species in addition to previously published data on Angiosperms [2]. To further address the distribution of BAP-like genes in other eukaryotes, we extended our dataset to include the representative genes encoded by non-plant bikonts and unikonts [3].

Plant-specific 4/1 polypeptide interacts with an endoplasmic reticulum protein related to human BAP31.

MAIN CONCLUSION: The plant-specific 4/1 protein interacts, both in yeast two-hybrid system and in vitro, and co-localizes in plant cells with plant BAP-like protein, the orthologue of human protein BAP31. In yeast two-hybrid system, we identified a number of Nicotiana benthamiana protein interactors of Nt-4/1, the protein known to affect systemic transport of potato spindle tuber viroid. For one of these interactors, an orthologue of human B-cell receptor-associated protein 31 (BAP31) termed plant BAP-like protein (PBL), the ability to interact with Nt-4/1 was studied in greater detail. Analyses of purified proteins expressed in bacterial cells carried out in vitro with the surface plasmon resonance (SPR) spectroscopy revealed that the N. tabacum PBL (NtPBL) was able to interact with Nt-4/1 with high-affinity, and that their complex can form at physiologically relevant concentrations of both proteins. Subcellular localization studies of 4/1-GFP and NtPBL-mRFP transiently co-expressed in plant cells revealed the co-localization of the two fusion proteins in endoplasmic reticulum-associated bodies, suggesting their interaction in vivo. The N-terminal region of the Nt-4/1 protein was found to be required for the specific subcellular targeting of the protein, presumably due to a predicted amphipathic helix mediating association of the Nt-4/1 protein with cell membranes. Additionally, this region was found to contain a trans-activator domain responsible for the Nt-4/1 ability to activate transcription of a reporter gene in yeast.

Phylogenetic and functional analyses of a plant protein related to human B-cell receptor-associated proteins.

Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem. Here, we have compared the sequences of PBL proteins and studied the biochemical properties of NtPBL. Analysis of a number of fully sequenced plant genomes revealed that PBL-encoding genes represent a small multigene family with up to six members per genome. Two conserved motifs were identified in the C-terminal region of PBL proteins. The NtPBL C-terminal hydrophilic region (NtPBL-C) was expressed in bacterial cells, purified, and used for analysis of its RNA binding properties in vitro. In gel shift experiments, NtPBL-C was found to bind several tested RNAs, showing the most efficient binding to microRNA precursors (pre-miRNA) and less efficient interaction with PSTVd. Mutational analysis suggested that NtPBL-C has a composite RNA-binding site, with two conserved lysine residues in the most C-terminal protein region being involved in binding of pre-miRNA but not PSTVd RNA. Virus-mediated transient expression of NtPBL-C in plants resulted in stunting and leaf malformation, developmental abnormalities similar to those described previously for blockage of miRNA biogenesis/function. We hypothesize that the NtPBL protein represents a previously undiscovered component of the miRNA pathway.