The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation

Abstract

In plants, the vascular tissue contains the enucleated sieve tubes facilitating long-distance transport of nutrients, hormones, and proteins. In addition, several mRNAs and small interfering RNAs/microRNAs were shown to be delivered via sieve tubes whose content is embodied by the phloem sap (PS). A number of these phloem transcripts are transported from source to sink tissues and function at targeted tissues. To gain additional insights into phloem-delivered RNAs and their potential role in signaling, we isolated and characterized PS RNA molecules distinct from microRNAs/small interfering RNAs with a size ranging from 30 to 90 bases. We detected a high number of full-length and phloem-specific fragments of noncoding RNAs such as tRNAs, ribosomal RNAs, and spliceosomal RNAs in the PS of pumpkin (Cucurbita maxima). In vitro assays show that small quantities of PS RNA molecules efficiently inhibit translation in an unspecific manner. Proof of concept that PS-specific tRNA fragments may interfere with ribosomal activity was obtained with artificially produced tRNA fragments. The results are discussed in terms of a functional role for long distance delivered noncoding PS RNAs.

Figure 1.

Size distribution of isolated small PS RNA and harvesting control. A, Denaturing PAGE of pumpkin. Shown are PS, petiole, stem, leaf, and petal small RNA labeled with [γ³²P]ATP. Indicated PS RNA fragments (asterisk) were eluted after PAGE, cloned, and sequenced. B, Contamination control by RT-PCR. PS RNA (approximately 0.5 μg) and leaf RNA (approximately 0.5 μg) were submitted to 30 RT-PCR cycles using primers amplifying phloem-specific CmPP16 mRNA or green tissue-specific Rubisco SSU mRNA. Note that with the PS RNA sample, no amplification of Rubisco SSU mRNA occurred. C, PS RNA harvesting and RNA processing control: in vitro-transcribed, [α³²P]-labeled Cm U4 snRNA and At U4 snRNA were added to PS droplets appearing on cut stem tissue, harvested, submitted to RNA isolation protocol, and analyzed via denaturing PAGE. −, RNA incubated with buffer; +, RNA added to PS droplet and re-isolated. Note that no processing or degradation of the RNA was observed.

Figure 2.

Size distribution and assignment of cloned small PS RNAs. A, The absolute number and size distribution of cloned PS RNA fragments compared to their appearance after denaturing PAGE. B, Pie graph showing the different classes and relative ratio of cloned sequences in percent according to sequence similarity. The cloned small PS RNAs could be grouped into six classes: rRNA, tRNA, snRNA, processing-related small RNA (proc. rel.), signal recognition particle RNA (SRP RNA), and ambiguous RNA with no meaningful similarity to know sequences.

Figure 3.

Northern assays confirm the presence of truncated rRNAs, snRNAs, and 7S signal recognition particle RNA in PS exudate. Approximately 5 μg PS RNA (PS) and pumpkin leaf RNA (LF) were transferred on nitrocellulose membranes after denaturing PAGE and incubated with specific oligonucleotide probes. Arrowheads, RNA fragments appearing specifically in the PS exudate.

Figure 4.

PS exudate tRNA fragments are processed outside the phloem. A, Northern-blot assays. Approximately 5 μg RNA from pumpkin PS, leaves (Cm), and Arabidopsis rosette leaves (At) were probed with labeled oligonucleotides hybridizing with specific tRNAs. Arrowheads indicate tRNA fragments detected exclusively in the PS. B, tRNA model indicating the number and size of tRNA fragments identified in the cDNA library. C, SDS-PAGE of leaf, stem, and PS protein extracts stained with Coomassie Brilliant Blue. D, Processing of in vitro-produced, [α³²P]ATP-labeled Met-tRNA by protein lysates as shown in C. Note that Met-tRNA processing was not observed with PS protein lysate. Arrowhead, Met-tRNA fragments formed after incubation with leaf or stem lysate. *, Shifted RNA band observed due to presence of phloem RNA-binding proteins.

Figure 5.

PS RNAs, not PS proteins, inhibit in vitro translation. Translation reactions were performed in the presence of [³⁵S]Met and protein production was determined by detecting labeled protein(s) after denaturing PAGE. A, Test of PS exudate translational activity. No translational activity of BMV RNA with PS lysate, weak translational activity after adding leaf RNA to WG; −, negative control avoiding RNA and positive control with BMV RNA (BMV) in WG extract. Note that the gel was overexposed to detect minor quantities of proteins produced in the translation assays. *, Unspecific background. B, PS exudate treated with RNase allows translation. PS exudate (5 μL) inhibited translation, whereas RNase-treated PS exudate allowed BMV RNA translation similar to the positive control without PS. C, Depletion of PS RNA. Compared to the + control with WG and BMV RNA, addition of PS exudate effectively inhibited BMV RNA translation. Increasing quantities of rRNase A (0.00007–0.014 units) added to the PS exudate resulted in an increased BMV RNA translation. Note that the added rRNase A was inactivated after incubation and did not affect BMV RNA translation (+ RNase control). Denatured PS RNA (RNA*) and PS proteins did not decrease BMV RNA translation. Arrowheads, BMV RNA-translated proteins with their expected sizes of 20 kD (coat protein), 32 kD (movement protein), 94 kD (RNA-dependent RNA polymerase), and 109 kD (methyltransferase/helicase). Bottom panel, Coomassie Brilliant Blue-stained protein-loading control. D, Statistical analysis of the relative translation efficiency in the presence of untreated PS (+PS), rRNase A-treated PS, and controls without PS (−PS). Number of repeated experiments, n ≥ 3. Error bars = sd of the means.

Figure 6.

Native PS RNA and truncated tRNAs inhibit translation. A, Comparison of small PS RNA size distribution among native isolated PS RNA, denatured PS RNA, and native re-isolated denatured PS RNA. *, RNA fragments appearing in different amounts. B, Inhibition of translation. Increasing amounts of native PS RNA, denatured PS RNA, native leaf RNA, denatured pumpkin leaf total RNA, and native yeast tRNA. Arrowheads, BMV RNA-translated proteins. C, Sequence specificity and translation inhibition by native PS RNA. FT, luciferase (Lucif.), and AtMPB2C RNA translation was inhibited by native (middle lanes), but not by denatured (denat.), PS RNA (right lanes). D, Native tRNA fragments produced by rRNase A inhibit translation. Top, Ethidium bromide agarose gel of yeast tRNA used in the translation assays. Bottom, SDS-PAGE of translated [³⁵S]Met-labeled BMV proteins. Note that yeast tRNA fragments inhibit BMV RNA translation, whereas full-length tRNA or fully degraded tRNA had no effect. B to D, Arrowheads indicate BMV RNA-translated proteins (for further details, see Fig. 5 legend). Lower panel, Coomassie Brilliant Blue-stained protein-loading control.