Identification of RNA silencing components in soybean and sorghum
© Liu et al.; licensee BioMed Central Ltd. 2014
Received: 1 July 2013
Accepted: 30 December 2013
Published: 4 January 2014
RNA silencing is a process triggered by 21–24 small RNAs to repress gene expression. Many organisms including plants use RNA silencing to regulate development and physiology, and to maintain genome stability. Plants possess two classes of small RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). The frameworks of miRNA and siRNA pathways have been established in the model plant, Arabidopsis thaliana (Arabidopsis).
Here we report the identification of putative genes that are required for the generation and function of miRNAs and siRNAs in soybean and sorghum, based on knowledge obtained from Arabidopsis. The gene families, including DCL, HEN1, SE, HYL1, HST, RDR, NRPD1, NRPD2/NRPE2, NRPE1, and AGO, were analyzed for gene structures, phylogenetic relationships, and protein motifs. The gene expression was validated using RNA-seq, expressed sequence tags (EST), and reverse transcription PCR (RT-PCR).
The identification of these components could provide not only insight into RNA silencing mechanism in soybean and sorghum but also basis for further investigation. All data are available at http://sysbio.unl.edu/.
Small RNAs, in particular, 20- to 24-nucleotide (nt) in length, belong to two classes: microRNAs (miRNAs) and short interfering RNAs (siRNAs). MiRNAs are regulators of gene expression and affect many biological processes, such as development and physiology in plants and animals [1–3]. Their dysregulation often causes developmental defects and diseases of plants and animals. MiRNAs are released as a duplex from an imperfect step-loop, which resides in the miRNA primary transcripts (pri-miRNA) [1–3]. SiRNAs are chemically indistinguishable with miRNAs but they originate from long perfect double-stranded RNAs (dsRNAs) [2, 3]. Plants encode several classes of siRNAs including siRNAs derived from repetitive DNAs (ra-siRNAs) and transacting siRNAs (ta-siRNAs) [2, 3]. Ra-siRNAs regulate gene expression at transcriptional levels by directing DNA methylation at homologs loci through a process named RNA-directed DNA methylation (RdDM) [2, 3]. In contrast, ta-siRNAs act like miRNAs to regulate gene expression at post-transcriptional levels . The framework of plant miRNA/siRNA biogenesis and function has been established in Arabidopsis thaliana (Arabidopsis); several different categories of genes are involved in the pathways for their generations and loading.
In Arabidopsis, the generation of miRNAs and siRNAs requires the DICER-LIKE proteins (DCL) . DCLs are the RNAase III enzymes that cut the dsRNAs to release ~ 22 nt RNA duplexes, which have 2 nt 3′ overhangs at each end . Arabidopsis encodes four DCLs: DCL1, DCL2, DCL3, and DCL4. DCL1, which associates with HYL1, a dsRNA binding protein, and SERRATE (SE), a zinc protein, cuts pri-miRNAs two times to release 21 nt miRNA duplex in nucleus [4–6]. DCL2 is responsible for 22 nt viral-derived siRNAs when plants are infected . DCL3 generates 24 nt ra-siRNAs and DCL4 produces 21 nt ta-siRNAs and some miRNAs [8–10]. The generation of both miRNAs and siRNAs also requires the single-stranded RNA (ssRNA)-binding proteins DAWDLE and TOUGH [11, 12]. After generation, miRNA and siRNA duplexes are 2′–O-methylated at 3′-terminal nucleotide by a dsRNA methylase HEN1 . The methylation protects miRNAs from degradation and 3′ untemplated uridine addition . The Arabidopsis HASTY (HST) gene is an ortholog of the human exportin 5 gene. After generation, miRNAs are exported to cytoplasm by HST-dependent or independent pathways , where they function. Interestingly, some components of miRNA biogenesis pathway are also targets of miRNAs. For example, in soybean, miR1515 can target DCL2 and leads to hypernodulation [16, 17].
RNA-dependent RNA polymerase (RDR) is another essential player for siRNA production. Among six RDRs in Arabidopsis, RDR2 converts ssRNAs generated from repetitive DNAs to precursor dsRNAs of ra-siRNAs , while RDR6 produces the ta-siRNA precursors . The generation of ra-siRNA also requires a plant specific DNA-dependent RNA polymerase IV (Pol IV) [19–21]. Pol IV is a Pol II-derived plant specific polymerase. It contains many identical subunits of Pol II , but the largest subunit NRPD1 and the second largest subunit NRPD2/NRPE2 of pol IV are paralogous of their counterparts in Pol II . Over 90% siRNAs require Pol IV for their production . Pol IV is thought to transcribe ssRNAs that serve as templates of RDR2 from RdDM target loci [19–21]. Another plant specific DNA dependent RNA polymerase V (Pol V) also plays crucial roles in the RdDM pathway [21, 24]. Pol V shares eight subunits with Pol IV including NRPD2/NRPE2 [22, 25], while NRPE1 (the largest subunit) and other three subunits are distinct from their counterparts in Pol IV [22, 25]. Pol V associates with RdDM target loci and produces ~200 nt non-coding transcripts from surrounding regions of some RdDM loci.
MiRNAs and siRNAs are loaded onto the ARGONAUTE (AGO) proteins, which performs target mRNA cleavage and/or translational inhibition, or directs chromatin modification such as DNA methylation . By recognizing the complementary sequences in the targets, miRNA,s and siRNAs guide AGO to silence specific genes . In general, there are multiple AGO genes in a plant species. Arabidopsis possesses 10 AGOs , based on sequence similarities, which are grouped into three clades: AGO1, AGO5 and AGO10 belong to the first clades; AGO2, AGO3 and AGO7 compose the second clades; and AGO4, AGO6, AGO8 and AGO9 are within the third clades . AGO1 associates with miRNAs and some siRNAs such as ta-siRNAs to cleave target mRNA and/or inhibit translation . AGO10 specifically sequesters miR166/165 from AGO1, which is essential for shoot apical meristem development [28, 29]. AGO7 binds miR390 to cleave the precursor RNA of ta-siRNAs . AGO4, AGO6, and AGO9 majorly bind 24 nt ra-siRNAs to direct DNA methylation , but seem to have different target preference [31–33]. It has been proposed that Pol V may recruit AGO4-siRNA complex to RdDM targets though its physical interaction with AGO4 and/or the interaction between its nascent transcripts and AGO4/6 associated siRNAs [34–36]. Recently, 19 and 18 AGOs were identified in rice and maize, respectively [37, 38].
In soybean and sorghum, our knowledge on RNA silencing mechanism is still poor. Taking advantage of available genome information and the conservation of RNA silencing components in different plant species, in this study, putative RNA silencing components, including DCL, HEN1, SE, HYL1, HST, RDR, NRPD1, NRPD2/NRPE2, NRPE1, and AGO, are identified in soybean and sorghum. The identification of these components could provide insight into RNA silencing mechanism in soybean and sorghum as well as basis for further investigation.
DCL genes in soybean and sorghum
Protein length (a)
HEN1, SE, HYL1, and HST
HEN, SE, HYL, and HST genes in soybean and sorghum
Protein length (a)
Soybean and sorghum genomes each encode three Arabidopsis SE homologs (Table 2). AtSEs have around 75% and 50-67% sequence similarities to GmSEs and SbSEs, respectively. Same as AtSEs, soybean and sorghum SEs possess an N-terminal unstructured region followed by an N-terminal domain containing several nuclear localization signals, a middle-domain, a core Zinc-finger domain, and a C-terminal unstructured region . Although similarities among GmSEs and among SbSEs are around 90%, their N-terminal unstructured regions (1–92 AA) are not conserved, which is consistent with the fact that the N-terminal unstructured region of a SE is not essential for its function in miRNA metabolism .
Both soybean and sorghum genomes encode two Arabidopsis HST homologs (Table 2). Although GmHSTa and b proteins are 79% similar to AtHST, they are 96% similar to each other. SbHSTa protein is 73% similar to AtHST, but SbHSTb shows only 59% similar to SbHSTa and 49% to AtHST. The low similarities of SbHSTb with SbHSTa and AtHST indicate that SbHSTb might be evolved into novel functions besides exporting miRNAs. Further research is deserved to conduct to test this hypothesis.
The dsRNA binding protein HYL1, which contains two dsRNA-binding domains at its N-terminus, is another essential component of miRNA biogenesis . Soybean genome encodes two HYL1 homologs that are 96% similar to each other, whereas sorghum encodes one HYL1 homolog (Table 2). GmHYL1a/b and SbHYL1 have more than 70% sequence identity with AtHYL1 at their N-terminal regions (~220 AA), which contains two dsRNA-binding domains. However, their C-terminal regions have no or little homology to that of AtHYL1. This is consistent with the fact that two dsRNA domains of HYL1 are essential and sufficient for its activity in miRNA biogenesis .
Pol IV and Pol V genes in soybean and sorghum
29724547 - 29732281
Protein length (a)
Soybean and sorghum Pol IV and Pol V
NRPD genes in soybean and sorghum
Protein length (a)
AGO genes in soybean and sorghum
Protein length (a)
DDH/H motifs in AGO genes
DCL is the essential component for miRNA and siRNA biogenesis . Although animals encode one DCL for the generation of both miRNAs and siRNAs, plants evolve four DCL groups . These DCLs have overlapping and diversified functions in miRNA and siRNA biogenesis . Both sorghum and soybean possess four DCL families, which further supports the notion that expansion of DCL family members in monocots and dicots happens after divergence between animal and plants . Sorghum has two DCL3 paralogs, DCL3a and DCL3b, which have low similarity to each other, whereas soybean encodes one DCL3. This result is consistent with the hypothesis that the DCL3 paralog in monocots was generated after divergence between monocots and dicots . OsDCL3a acts in non-canonical long miRNA biogenesis and 24 ra-siRNA biogenesis, whereas OsDCL3b functions in phased 24-nt siRNA biogenesis, indicating that the function of DCL3 paralogs is diversified . Because of the high similarities of SbDCL3a to OsDCL3a and SbDCL3b to OsDCL3b, SbDCL3a/b most likely have different functions in the small RNA pathway.
In Arabidopsis, DCL1, SE, TOUGH and HYL1 form a complex to process pri-miRNA in nucleus to generate miRNA duplex that are methylated by HEN1 and exported into cytoplasm by HST [4–6, 12, 13, 15]. The identification of DCL1, HYL1, SE, HEN1, and HST homologs in sorghum and soybean suggests that the biogenesis processes of miRNAs in them are similar to that of Arabidopsis. It is noted that in sorghum, the paralogs of HYL1, SE, HEN1, and HST are less similar to each other, but each has a closely related homolog in rice. This indicates that the duplication may occur before divergence between rice and sorghum about 50–70 million years ago . However, one can note that SEs in both soybean and sorghum have three paralogs each, which is more than other components in soybean/sorghum and SE in Arabidopsis do. This indicates the selective duplication for SEs in soybean and sorghum, besides whole genome duplication.
RDR is essential for siRNA biogenesis as well . Studies from Arabidopsis, rice, and maize have shown that plants possesses four groups of RDRs: RDR1, RDR2, RDR3 and RDR6. RDR2 from Arabidopsis and maize (MOP2), RDR6 from Arabidopsis and rice are required for ra-siRNA and ta-siRNA biogenesis, respectively [45, 46]. Recently, it was shown that RDR6 acts redundantly with RDR1 in viral-derived siRNA biogenesis . The function of RDR3 family is currently unknown yet. Corresponding RDR1, RDR2, RDR3, and RDR6 homologs for both soybean and sorghum are identified, which further supports the notion that the RDR gene family in plants is derived from a common ancestor.
The putative largest subunit and the second largest subunit of Pol IV and PolV, which are required for ra-siRNA-mediated DNA methylation, are discovered from soybean and sorghum. This agrees with the notion that Pol V and Pol IV are plant specific polymerases. In maize, lack of Pol IV and Pol V causes development defects , whereas in Arabidopsis, the nrpd and nrpe mutants appear to grow normally. It is interesting to further test whether Pol IV and Pol V are necessary for the development of soybean and sorghum.
AGO is the effector protein for small RNA-mediated silencing . It is proposed that both plants and animals encode multiple AGOs to meet the diversified functions of small RNA silencing . Like rice, maize, and Arabidopsis, both soybean and sorghum encode three subfamilies of AGO proteins, indicating that small RNA functions are conserved in higher plants. Soybean encodes seven AGO10 paralogs. Among of them, GmAGO10a/b/c share high similarity to each other, while GmAGO10d/e/f/g are clustered. The similarity of these two groups of GmAGO10 is relatively low, which indicates that their functions might be different. They might regulate the functions of different miRNAs. In Arabidopsis, AGO10 has been shown to regulate the function of miR166/165 [28, 29].
The identification of these putative RNA silencing components would give insight on small RNA pathways in soybean and sorghum. However, the exact function and contribution of individual component of RNA silencing machinery needs to be further examined because their functions may be diverse among different plant species.
Small RNA-mediated gene silencing is an important mechanism to regulate gene expression and genome stability in plants. The available sorghum and soybean genome information enable the identification of components that may involve in small-RNA mediated gene silencing in soybean and sorghum [59, 60]. The gene families, including DCL, HEN1, SE, HYL1, HST, RDR, NRPD1, NRPD2/NRPE2, NRPE1, and AGO, in soybean and sorghum were identified. RNA-seq, EST and RT-PCR analysis confirmed the expression of these candidate genes. In soybean, the similarities among paralogs are very high, which is consistent with the hypothesis that there have been 1–2 rounds of genome duplication in soybean since the separation of homolog sequences between soybean and Arabidopsis approximately 90 million years ago . Based on the knowledge of their counterparts in Arabidopsis, putative functions to these genes are annotated.
Genome sequence data
We collected soybean (Gmax 189) and sorghum (v1.4) genome sequences from Phytozome (v9.0) (http://www.phytozome.net/), and Arabidopsis sequences from TAIR (10) (http://www.arabidopsis.org/). The total numbers of genes are 55787, 35386, and 29448 for soybeans, sorghum, and Arabidopsis, respectively.
Identification of miRNA components
HMM analysis was used to search for DCL, AGO, and RDR genes encoded in the soybean and sorghum genomes, besides searching homolog in Arabidopsis with TBLASTN. DCL proteins have domains of DExD-helicase, helicase-C, Duf283, PAZ, RNase III, and double-stranded RNA-binding (dsRB). AGOs have PAZ, MID, and PIWI domains. RDRs have a conserved RDRP domain. The HMM profiles of domains in DCL, AGO and RDR families are obtained from the Pfam database. With the HMM profiles, the corresponding conserved sequences of DCL, AGO, and RDR proteins are extracted by HMMER . These conserved sequences are adapted to search for all predicted DCL, AGO and RDR genes. Protein sequences of all candidate genes were also aligned against Arabidopsis genome with BLASTP program (cutoff E-value = 0.001). The other genes, HEN1, SE, HYL1, and HST, which have only one gene in Arabidopsis, were screened against soybean and sorghum genomes with TBLASTN program (cutoff E-value = 0.001) to find the candidate genes.
Clustal-W was used for multiple sequence alignments. Phylogenetic analysis was performed with the PhyML and MEGA v5.0 programs by the maximum-likelihood method with 500 bootstrap replicates.
RNA-seq data analysis
RNA-seq data for soybean and sorghum were obtained from SRA (http://www.ncbi.nlm.nih.gov/Traces/sra/), and the accession numbers of these RNA-seq data are SRX062333 (floral bud), SRX113962 (cotyledons), and SRX265552 (seeds) for soybean and SRX080311 (root), SRX080321 (shoot), SRX080322 (shoot), SRX080323 (shoot), SRX099022 (early inflorescence), and SRX099184 (embryo) for sorghum. After preprocessing the RNA-seq data, the short reads were mapped against the G. max 189 genome and S. bicolor v1.4 genome sequences using Tophat (v1.3.2) , allowing up to two mismatches. The numbers of reads in genes were counted by HTSeq-count tool (Anders, 2010)  with the “union” resolution mode, and they are normalized with scaling the total count of mapped reads to 10 million reads. For each gene, the numbers of mapped reads per kilobase of exon per million mapped reads (RPKM) is shown as well.
EST expression analysis
To estimate the expression profiles, all miRNA components are searched against the dbEST database  (http://www.ncbi.nlm.nih.gov/dbEST) and PlantGDB  (http://www.plantgdb.org) with MEGABLAST (cutoff E-value = 10-10).
Total RNAs from inflorescences of soybean or sorghum was extracted as described in the work of Yu et al. . After treatment with DNase I, 5 μg RNA was reverse transcribed (RT) by the Superscript III reverse transcriptase (Invitrogen) using an oligo-T18 primer to generate cDNAs at 50°C for 1 hour. The resulting cDNAs was used as templates to perform PCR amplification with primers listed in Additional file 4: Table S3. PCR was performed for 32 cycles (94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds). Total RNAs were extracted from inflorescences of soybean or sorghum. Reverse transcription was performed using an Oligo-T primer. The amplification of UBIQUITIN 5 (UBQ5) was used as a loading control.
The work is supported by funding under CZ’s startup funds from University of Nebraska, Lincoln, NE, USA and BY’s grant from Nebraska Soybean Board, NE, USA.
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