Phloem transport of structured RNAs: A widening repertoire of trafficking signals and protein factors.

The conducting sieve tubes of the phloem consist of sieve elements (SEs), which are enucleate cells incapable of transcription and translation. Nevertheless, SEs contain a large variety of RNAs, and long-distance RNA trafficking via the phloem has been documented. The phloem transport of certain RNAs, as well as the further unloading of these RNAs at target tissues, is essential for plant individual development and responses to environmental cues. The translocation of such RNAs via the phloem is believed to be directed by RNA structural elements serving as phloem transport signals (PTSs), which are recognized by proteins that direct the PTS-containing RNAs into the phloem translocation pathway. The ability of phloem transport has been reported for several classes of structured RNAs including viroids, genuine tRNAs, mRNAs with tRNA sequences embedded into mRNA untranslated regions, tRNA-like structures in the genomic RNAs of plant viruses, and micro-RNA (miRNA) precursors (pri-miRNA). Here, three distinct types of such RNAs are discussed, along with the proteins that may specifically interact with these structures in the phloem. Three-dimensional (3D) motifs, which are characteristic of imperfect RNA duplexes, are discussed as elements of phloem-mobile structured RNAs specifically recognized by proteins involved in phloem transport, thus serving as PTSs.

Data on occurrence of miRNA precursors in the Cucurbita maxima phloem sap.

The phloem sieve elements (SEs), enucleate cells, contain RNAs, which are imported from surrounding tissues and cells, mostly companion cells tightly associated with SEs, and transported via the phloem over the whole plant body. The RNA phloem transport is essential for plant individual development and responses to environmental cues. Recently, we identified primary miRNA (pri-miRNA) sequences in de novo assembled transcriptome of Cucurbita maxima phloem sap and reported 11 most abundant pri-miRNAs [1]. Here, we provide the output of this analysis in complete detail. For the full set of pri-miRNAs identified in the C. maxima phloem sap transcriptome, data on relative abundance are provided along with annotated sequence data.

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.

RNA phloem transport mediated by pre-miRNA and viral tRNA-like structures.

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3'-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.