Small RNAome sequencing delineates the small RNA landscape of pluripotent adult stem cells in the planarian Schmidtea mediterranea

Abstract

Small noncoding RNAs play a pivotal role in the regulation of gene expression, and are key regulators of animal development. Freshwater planarian exhibits an extraordinary ability to regenerate any missing body parts, representing an emerging model for studying mechanism underlying stem cell regulation and tissue regeneration. Here, we utilized next-generation sequencing (NGS) to identify small RNAs that are expressed in planarian adult stem cells, and are implicated in tissue regeneration. We profiled microRNAs (miRNAs), piwi-interacting RNA (piRNAs), small rDNA-derived RNAs (srRNAs) and endogenous interfering RNAs (endo-siRNAs) population from size 18-30 nt, measured the expression of 244 conserved miRNAs, and identified 41 novel miRNAs and 64 novel endo-siRNAs. Expression profiling analyses revealed that most piRNAs and srRNAs are up-regulated during regeneration, and that the most abundantly expressed srRNAs are from 5.8s and 28s rRNA. Furthermore, a target prediction method was adopted to investigate the anti-correlation of small RNAs and mRNA expression. We built up a gene regulatory network based on the genes that are targeted by dynamically changed small RNAs. These results expand the known small RNA repertoire in planarian, and provide valuable insights and a rich resource for understanding the small RNAs landscape in stem cell-mediated regeneration.

Fig. 1

Stem cell depletion by HDAC1 RNAi and irradiation. (A) Immunostaining of stem cell marker expression with PIWI-1 and HP-1 antibodies after gene known-down or irradiation. (B) Immunostaining of mitotic marker H3S10P after gene known-down. (C) The number of neoblasts decreased following irradiation or HDAC1 RNAi. Error bars represent SD; ^⁎⁎⁎equals p < 0.0001; significance determined with Student's t-test.

Fig. 2

Length distribution of small RNAs. (A) Redundant small RNAs in sexual intact and amputation samples. (B) Redundant small RNAs in asexual intact, amputation, HDAC1 KD and irradiation samples. (C) Unique small RNAs in sexual intact and amputation samples. (D) Unique small RNAs in asexual intact, amputation, HDAC1 KD and irradiation samples.

Fig. 3

Heatmap of total miRNAs in sexual intact, amputation; asexual intact, HDAC1 KD, irradiation and amputation samples.

Fig. 4

Scatter plots of miRNAs in different samples. (A) Sexual amputation compared with intact sample. (B) Asexual amputation compared with intact sample. (C) Asexual HDAC1 knockdown compared with intact sample. (D) Asexual irradiation compared with intact sample.

Fig. 5

Scatter plots of piRNAs in different samples. (A) Sexual amputation compared with intact sample. (B) Asexual amputation compared with intact sample. (C) Asexual HDAC1 knockdown compared with intact sample. (D) Asexual irradiation compared with intact sample.

Fig. 6

Positions of srRNAs in different type of ribosomal RNAs. (A) sme5.8s. (B) sme28s. (C) gti28s. (D)nve18s.

Fig. 7

Scatter plots of srRNAs in different samples. (A) Sexual amputation compared with intact sample. (B) Asexual amputation compared with intact sample. (C) Asexual HDAC1 knockdown compared with intact sample. (D) Asexual irradiation compared with intact sample.

Fig. 8

The secondary structure of endogenous siRNA. (A) siRNA-0001 on transcriptome SRR955511.11038203. (B) siRNA-0002, 0008 on transcriptome SRR955099.8344711. (C) siRNAs-0003, 0011 on transcriptome SRR955099.157334. (D) siRNA-0004, 0010 on transcriptome SRR955099.6371659. (E) siRNA-0004, 0060 on transcriptome SRR955099.5306530.