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Identification of small non-coding RNAs responsive to ZnSO4 stress in gonad of sea urchin (Strongylocentrotus nudus).

Byline: Zhenlin Wei

Abstract

In this research a large number of putative siRNAs and piRNAs were identified in male gonad of sea urchin and subjected to microarray assay to compare their expressional profile between normal and 0.1mM Zins treated gonad. The results showed 24 up- and 57 down-expressed siRNAs and piRNAs in stressed gonad. Subsequently functional annotation showed 5 down- expressed ncRNAs (3 siRNAs and 2 piRNAs) have 2 types of retrotransposons 2 types of simple repeat motifs and a few mRNAs and ESTs as their putative targets. More so the majority putative siRNA and piRNAs that differently expressed under Zinc stress was neither mapped to selfish elements nor transcribed from repeat sequences which were similar to their mammal counterparties. Results revealed the biological significance of small non-coding RNAs in responding to heavy metal stress. Copyright 2014 Friends Science Publishers.

Keywords: Small ncRNAs; Solexa sequencing; Microarray assay; Zinc stress; Sea urchin.

IntroductionMetal-working industries are developed with a surprising progress as the problem of heavy metal pollution on sea environment has become increasingly attractive. There are growing studies focus on the heavy metal pollutants in the marine environment and on the accumulation in tissues of various organisms (Zehra et al. 2003). Exposure For decades sea urchins have been used to sensor seawater pollutant and to elucidate the mechanism of heavy metal toxicity (Wei et al. 2011) because urchin is a convenient system since many aspects of metal homeostasis and stress resistance are conserved between urchins and humans.It has been documented that environmental pollutants have been shown to interfere with reproductive function in animals. Currently there is great interest in characterizing the functions of the different classes of ncRNAs and their relevance to heavy metal treatmentssince small non-coding RNAs were uncovered as key regulators controlling variousstress responses (Yao et al. 2010). Although sea urchin iswidely used for evaluating biological effects of contaminants in marine environment there are only few studies investigating the effects of chemicals on gonad of sea urchins.Recently we carried out a deep sequencing applied to sea urchin Strongylocentrotus nudus in a small RNA library isolated from male and female gonad and found 36 miRNAs significantly varied between control and ZnSO4 stressed samples (Wei et al. 2011) providing the information about the roles of small non-coding RNAs in resistance to abiotic stress. However the potential functions of other classes of ncRNAs such as siRNAs and piRNAs in sea urchin on responding to heavy metal stress is absent yet although thesirnas have been verified to participate in stress adaptation (Simmons et al. 2009). Whether the siRNAs and piRNAs participated in cellular response to heavy metal in sea urchin gonad remain concerning.

Materials and Methods

Sample Collection and ZnSO4 Treatment

Sea urchin (Strongylocentrotus nudus) with about 5 cm in diameters was collected in the coast of Dalian liaoning province China. The animals were acclimated in laboratory with filters natural seawater for a week prior to RNA extraction prior to treatment with ZnSO4 at 0.1 mM for 12 h.

RNA Isolation Solexa Sequencing and Microarray Assay

Total RNA of S. nudus was extracted from male gonad usingTrizol reagent (Invitrogen CA USA) and approximately 20g of total RNA was used for solexa sequencing (Wei et al.2011). After masking of adaptor sequences and removal of contaminated reads the clean reads were filtered for siRNA and piRNA identification with ACGT101-miR-v3.5 package (LC Sciences Houston USA). Total 202 siRNAs and 153 piRNAs were selected to design probes (in triplicate) and subject to expressional profile analysis between normal and ZnSO4 treated male gonad usingParaflo(TM) small ncRNA microarray (LC SciencesHouston USA) assay and data analyzed (Gao et al. 2004).Target Prediction and AnnotationThe siRNA and piRNA sequences were used as query to search S. purpuratus mRNA and repeat sequences to perform targets prediction using BLASTn with -E as 1e-0.0001 (Wang et al. 2011). The targets were then subjected toBLASTx and KEGG searching.

Results

Identification of siRNA and piRNA Using SolexaSequencing

Sequencing of small RNAs generated 4966572 reads then the reads matched to rRNA tRNA sno/snRNA mRNA and miRNAs were discarded. From remaining 11433100 reads total putative 4968 siRNAs were identified according to the criteria that these sequences do not derived from hairpin structures and are not related to other ncRNAs deposited in Rfam and NT databases. Of these siRNAs 2584 sequences complementary to S. purpuratus mRNA ESTs and repeat elements were recognized as endo-siRNAs. Majority (62%) of the small RNAs were 16 to 26 nt in size appearing typical feature for Dicer-derived products. The length distribution and 5' end bias is illustrated in Fig. 1C. The average copy number is 184 while total copy number is 1274728.Furthermore two groups of putative piRNA like RNAs were identified based on their base composition and genome mapping information. The first group comprised 12201 sequences with U" at the first position of 5'end (termed as5U RNAs) while the second group comprised 851 sequences with A" at the position 10 from 5'end and with10 bp overlapped to the sequences in the first group (termed as10A RNAs) similar to that observed by Aravin et al (2007).

Microarray Analysis of siRNAs and piRNAs in Normal and Zinc-stressed Male GonadResults of microarray assay showed drastically decreasing of expressions of siRNA and piRNA under Zinc stress resulting in losing signals of 77 small RNAs including 70piRNAs and 7 siRNAs under stress. Out of 81 ncRNAs varied significantly between control and Zinc stressed male gonad 57 of them were down-regulated up to 4.09 times that of control (Fig. 1A). Interestingly among the top 20 abundant small RNAs with signals from 24380 to 16500 (control) 14 ncRNAs showed significant increasing of signal under Zinc stress and siRNA92 give the maximum increasing from 20090 to 89362. In contrast among 56 small RNAs with array signal from 12000 to 3000 45 showed obvious decreasing of signals (Fig. 1A) implying that small ncRNAs appeared different expressional profiles.

Functional Annotation of Putative Targets

Recent findings show that siRNA could target either repeat elements or mRNAs while piRNAs have shown a role in the silencing of DNA transposons and retrotransposons in several animal models (Aravin et al. 2007) these allow us to search their complementary RNAs. According to the methods described by Wang et al. (2011) BLASTn was used to find potential targets of putative siRNAs and piRNAs. The results revealed 3 siRNAs and 2 piRNAs highly complementary to sequences of 2 types of simple repeat

Table 1: siRNAs complement to sea urchin mRNAs

ncRNA###Express-###Complemented Complemented Functional annotation

ID###ional###sequence###regions

###variation

wsir21 -1.15###M20117###61-85###CHK1

wsir28 -1.09###M20117###186-210###CHK1

wsir124 -2.26###M20117###83-106###CHK1

wsir23 -1.98###M20118###270-292###Similar to

###DAPPUDDRAFT_67127

wsir52###-1.65###M20117###63-83###CHK1

wsir40###-1.13###M20118###30-47###Similar to

###DAPPUDDRAFT_67127

wsir83###1.63###AY465426###633-650###PLC

motifs and two types of transposons including SINE2-1_SP and SPRP1. These 5 ncRNAs were down-regulated under Zn stress suggesting an attenuated silencing for these TEs.Moreover BLASTn against sea urchin mRNAdatabase showed 2 mRNA sequences were complemented by these them phospholipase C delta (PLCd) was targeted by siRNA83 which up expressed in Zinc stressed gonad. PLCd was functional mapped onto KEGG 00562 04020 and04070 indicating a key role in IP3-dependent Ca2+ signaling system. Inhibition of PLC has been proved as one of toxiceffects of Hg2+ and Cu2+ on Mytilus galloprovincialis Lam (Panfoli et al. 2000). In other instance Checkpoints kinase 1 (CHK1) was intensively targeted by 4 siRNAs that all down expressed under stress conditions (Table 1) indicating a decreased expressional controlling of chk1. Taking intoaccount the CHK1 is one of the key proteins responding to stress this result maybe uncover a novel relationship between Zinc stress and small ncRNAs regulations.Furthermore these siRNAs were BLASTn against EST database of sea urchin which showed 272 ESTs to be putative targets of siRNAs. Then these ESTs were assembled and functionally annotated. Of these 13 ESTs were mapped to GO database with cutoff lower than 1e-10. In the context of semantic similarity of GO terms (Fig. 1B) the functions of targeted ESTs fell into two clusters one comprised protein binding (GO:0051082 GO:0003779 and GO:0005519) another included nucleoid acid binding activities (GO:0003690 GO:0003677 and GO:0003723). These data provided new idea on how can small non-coding RNAs participate in responding to heavy metal stress in sea urchin.

DiscussionPrevious research has evidenced that sea urchin evolved a conserved pathway contributing to biogenesis of small non- coding RNAs such like miRNAs siRNAs and piRNAs (Wei et al. 2012). In addition to discovering of miRNAs and piRNAs (Wei et al. 2011; Wei et al. 2012) this research identified 2584 siRNAs in the male gonad of sea urchin thereafter conduce to elucidate the biological roles of these small RNAs both in development and stress resistance.Zinc treatment remarkably changed the expressional profiles of tested siRNAs and piRNAs indicating a putative roles of these RNAs in responding to stress resulting in either up-expression or down expression for each ncRNA. Furthermore BLASTn searching revealed 2 types of transposons as putative targets of 5 down regulated ncRNAs suggesting that these repeat sequences would be more activity under Zinc stress. Typically increasing transposons activity is one reason for gonadal dysgenesis (Kale et al.2005) therefore it is likely that attenuated silencing of TEs is served as a novel inducer for developmental abnormal of sea urchin under heavy metal stress (Kale et al. 2005). In consist with this hypothesis Farkash et al. (2006) found that abiotic stress increased endonuclease-dependent L1 retrotransposition. Therefore this results indicated that small ncRNAs are newly emerged regulators concerning to heavy stress by means of controlling chromosome remodeling in sea urchin gonad similar to other animal model (Mizutani et al. 2012).Similar to previous research the 5U piRNAs were rare complemented to TEs well consisting with the notion that small RNAs with function to silence gene expression by multiple mechanisms (Yao et al. 2010). In adult testis of mammals the piRNAs have no known function and in mouse testes the promoter regions of particular retrotransposons are methylated through cooperating by piRNA and several proteins (Cowley and Oakey 2013). Considering the observations that urchin piRNAs were less come from repeat-rich regions and the identification of cognate of DNMT3B in urchin genome (Sp-Dnmt3A and SPU_024347) we suggested that the piRNA identified in this research are functionally similar to their mammalcounterparties. On other hand only 355 small ncRNAs were tested in this research while thousands of small ncRNAs were remained for analysis in further research. Therefore it is likely that the small ncRNAs identified in this research have more complex functions responding to Zn stress such like methylating promoter regions of particular retrotransposons (Shpiz et al. 2011).In crux out of 355 siRNAs and piRNAs 81 siRNAs and piRNAs expressed significatively different under Zinc stress. Subsequently bioinformatical analysis showed these81 ncRNAs were targeted to 2 types of repeat sequences and to a few number of mRNAs and ESTs. Data revealed the biological significance of small ncRNAs on heavy metal stress in male gonad of sea urchin.

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (Grant No. 31272704)

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Author:Wei, Zhenlin
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Aug 31, 2014
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