The sRNAome mining revealed existence of unique signature small RNAs derived from 5.8SrRNA from Piper nigrum and other plant lineages

Abstract

Small RNAs derived from ribosomal RNAs (srRNAs) are rarely explored in the high-throughput data of plant systems. Here, we analyzed srRNAs from the deep-sequenced small RNA libraries of Piper nigrum, a unique magnoliid plant. The 5' end of the putative long form of 5.8S rRNA (5.8S_LrRNA) was identified as the site for biogenesis of highly abundant srRNAs that are unique among the Piperaceae family of plants. A subsequent comparative analysis of the ninety-seven sRNAomes of diverse plants successfully uncovered the abundant existence and precise cleavage of unique rRF signature small RNAs upstream of a novel 5' consensus sequence of the 5.8S rRNA. The major cleavage process mapped identically among the different tissues of the same plant. The differential expression and cleavage of 5'5.8S srRNAs in Phytophthora capsici infected P. nigrum tissues indicated the critical biological functions of these srRNAs during stress response. The non-canonical short hairpin precursor structure, the association with Argonaute proteins, and the potential targets of 5'5.8S srRNAs reinforced their regulatory role in the RNAi pathway in plants. In addition, this novel lineage specific small RNAs may have tremendous biological potential in the taxonomic profiling of plants.

Figure 1. The cleavage points during the processing of 5.8S rRNA from pre-ribosomal RNA.

Figure 2

Distribution and abundance pattern of ribosomal RNA derived small RNAs in Piper nigrum small RNA libraries (A) Distribution of unique srRNA candidates against black pepper rRNA. (B) Abundant srRNA from Pn_CL (upto100 rpm) mapped against rRNA. Most of the abundant reads were mapped at the 5.8S rRNA region. (C) The proportion of 5.8S rRFs in the Pn_CL, Pn_IL and Pn_IR libraries. (D) The length categorisation of 5.8S rRFs among the three libraries.

Figure 3. Abundance analysis of 5.8S rRFs.

(A) The 5′5.8S rRFs were the most abundant category of srRNAs in Piper nigrum. srRNAs with read numbers > 10 were analyzed. (B) The 5′ conserved sequences from different species of Piper. (C) The mapped srRNAs to 5.8S rRNA.

Figure 4. Multiple sequence alignment of rRNAs from different plant species.

GACTCTCGGCAACGGATAT is the 5′5.8S rRF mapped 5′ motif found in all the lineages of spermatophyte plants.

Figure 5

Analysis of isoforms and cleavage pattern of 5′5.8S rRFs from black pepper (A) Sequence logo generated from the different isoforms of 5′5.8S rRFs. (B) The cleavage pattern of 5.8S rRFs assessed upstream of the GACUCUC consensus (C) The length distribution of 5′5.8S rRFs.

Figure 6. Unique sequence variation at the 5′ ends of 5.8S rRNAs and the prominent 5.8S rRFs in plants.

(A) Sequence diversity at the 5′ termini of 5.8S rRNA in different plant species (B) Multiple sequence alignment of 5′5.8S rRF from different plant lineages. The most predominant 5′5.8S rRFs from each plant species were aligned using ClustalW. (tae: T.aestivum, sbi: S. bicolor, sit: S. italica, hvu: H. vulgare; osa: O. sativa; pvi: P. virgatum, zma: Z. mays, mgi: M. giganteus, mac: M. acuminata, nad: N. advena, atr: A. trichopoda, afi: A. fimbriata, pam: P. americana, pni: P. nigrum, vvi: V. vinifera, cpa: C. papaya, csi: C. sinensis, gar: G. arboretum, ptr: P. trichocarpa, cma: C. maxima, ath: A. thaliana, ahy: A. hypogaea, mtr: M. truncatula, gma:G. max, can: C. annuum, sly: S. lycopersicum, stu: Solanum tuberosum, nta: N.tabacum, mgu: M. guttattus, sla: S. latifolia, cru: C. rumphiis, gbi: G. biloba, pab: P. abies, mqu: Marsilea quadrifolia.

Figure 7. Association of 5′5.8S rRFs with different Argonaute proteins.

5′5.8S rRFs identified from AGO immuno-precipitated sRNA libraries of A. thaliana. (A) AGO1 (B) AGO4 (C) AGO2 & AGO7 (D) AGO6 & AGO9 (E) 5′5.8S rRFs identified from different homologs of AGO1 immuno-precipitated sRNA libraries of O. sativa. The sRNA data (GSM707682, GSM707686, GSM304284, GSM415789 and GSM415791) from the studies of Wang et al., 2011, Montgomery et al., 2008 and Havecker et al., 2010 were analysed and the total read counts of 5′5.8S rRFs was represented as the normalized read count (RPM) from the corresponding sRNA library. (F) 5′5.8S rRFs detected from the Ago1–5 mutants.

Figure 8. The predicted secondary structure of 5.8SrRNA of black pepper.

The secondary structure of (A) P. nigrum and (B) A. thaliana 5.8S rRNA as predicted from the RNAFold. (C) The non-canonical short hairpin precursor of 5′5.8S rRFs. The 5′5.8S rRF was highlighted in red color. The next abundant rRF identified from the sRNA library was highlighted in blue in the opposite strand.

Figure 9. Involvement of Dicer-like activity on the biogenesis of srRNAs.

The expression of 5′5.8S rRFs detected from the dcl-1 and dcl-234mutant Arabidopsis plants.

Figure 10. The expression of 5′5.8S rRF variants in the Pn_IL and Pn_IR libraries compared to the control leaf library (Pn_CL).

Figure 11. Experimental mapping of 5′5.8S rRF mediated cleavage on the predicted target.

(A) Cleavage was mapped on the predicted rRF binding site in the mRNA transcript encoding 40S ribosomal protein S13 from black pepper by modified 5′RLM RACE experiments. The red arrow indicates mapped cleavage sites and the number indicates the frequency of clones from RACE experiments (B) 5′5.8S rRF cleavage sites on the CL1091.Contig 2 coding for 40S ribosomal protein S13.

Figure 12. The 5.8S rRF mediated cleavage on the predicted targets detected from the Arabidopsis degradome data.

The cleavage was detected on the rRF aligned position on the target mRNAs of ribosomal L5P family protein, at the L5 domain.