- Research article
- Open Access
Beyond cleaved small RNA targets: unraveling the complexity of plant RNA degradome data
© Hou et al.; licensee BioMed Central Ltd. 2014
- Received: 9 August 2013
- Accepted: 6 January 2014
- Published: 10 January 2014
Degradation is essential for RNA maturation, turnover, and quality control. RNA degradome sequencing that integrates a modified 5′-rapid amplification of cDNA ends protocol with next-generation sequencing technologies is a high-throughput approach for profiling the 5′-end of uncapped RNA fragments on a genome-wide scale. The primary application of degradome sequencing has been to identify the truncated transcripts that result from endonucleolytic cleavage guided by microRNAs or small interfering RNAs. As many pathways are involved in RNA degradation, degradome data should contain other RNA species besides the cleavage remnants of small RNA targets. Nevertheless, no systematic approaches have been established to explore the hidden complexity of plant degradome.
Through analyzing Arabidopsis and rice RNA degradome data, we recovered 11 short motifs adjacent to predominant and abundant uncapped 5′-ends. Uncapped ends associated with several of these short motifs were more prevalent than those targeted by most miRNA families especially in the 3′ untranslated region of transcripts. Through genome-wide analysis, five motifs showed preferential accumulation of uncapped 5′-ends at the same position in Arabidopsis and rice. Moreover, the association of uncapped 5′-ends with a CA-repeat motif and a motif recognized by Pumilio/Fem-3 mRNA binding factor (PUF) proteins was also found in non-plant species, suggesting that common mechanisms are present across species. Based on these motifs, potential sources of RNA ends that constitute degradome data were proposed and further examined. The 5′-end of small nucleolar RNAs could be precisely captured by degradome sequencing. Position-specific enrichment of uncapped 5′-ends was seen upstream of motifs recognized by several RNA binding proteins especially for the binding site of PUF proteins. False uncapped 5′-ends produced from capped transcripts through non-specific PCR amplification were common artifacts among degradome datasets.
The complexity of plant RNA degradome data revealed in this study may contribute to the alternative applications of degradome in RNA research.
- RNA degradation
- RNA motif
- RNA-binding protein
- Sequencing artifact
Degradation plays vital roles in RNA maturation, turnover, and quality control. Almost all RNA species are transcribed longer before becoming functional forms and require the removal of extra sequences in the termini (5′ or 3′ processing) or internal regions (splicing). Mature 5′ RNA ends generally possess a triphosphate or a 7-methylguanosine cap, whereas mature 3′ RNA ends possess a poly(A) tail or a stem-loop structure. Loss of these specific features stimulates RNA turnover . Defective RNAs containing a premature stop codon, lacking an in-frame stop codon or carrying stalled ribosomes are eliminated by mRNA-surveillance pathways [2–5]. RNA degradation can proceed from the 5′-end, the 3′-end, or internally with 5′-to-3′ exoribonucleases, 3′-to-5′ exoribonucleases, and endoribonuclease, respectively. Maturation of ribosomal RNAs (rRNAs), transfer RNAs, small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) relies on the delicate cooperation of exoribonucleases and endoribonuclease. Cis-elements on mRNAs can trigger endonucleolytic cleavage or deadenylation and therefore destabilize RNA. The exosome is the major component in versatile RNA maturation and surveillance pathways . Some exoribonucleases have dual functions, and can degrade entire transcripts for some RNA species and define the termini of mature RNAs for other RNA species. For instance, the yeast 5′-to-3′ exoribonuclease Rat1 participates in the degradation of unspliced pre-mRNAs as well as the formation of snoRNA 5′-ends [7, 8].
Small regulatory RNAs (20–24 nt) such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) can initiate endonucleolytic cleavage in the middle of highly complementary target sites on long transcripts . Small RNA-guided cleavage is mediated by Argonaute proteins which possess small RNA binding domains and endonuclease domains . The 3′ cleavage remnant of some plant miRNA targets is the substrate of a 5′-to-3′ exoribonuclease, XRN4/EIN5 . Specific cleavage sites initiated by small RNAs are frequently validated using a modified 5′-rapid amplification of cDNA ends (5′ RACE) protocol that skips enzyme treatment for the removal of the 5′ phosphate and the capping structure . With this modification, 5′ RNA adaptors can only ligate to RNA molecules without a cap structure but with a monophosphate at the 5′-end which are the typical products of small RNA-guided cleavage, thus preventing sequencing of full-length mRNAs with a cap structure. Advances in high-throughput sequencing technologies have enabled genome-wide surveys of uncapped RNA molecules and parallel validation of numerous small RNA targets. High-throughput methods for profiling uncapped RNA termini have been established independently by several groups and are known variously as degradome sequencing, parallel analysis of RNA ends (PARE) and genome-wide mapping of uncapped transcripts (GMUCT) [13–15]. The three approaches all start with the enrichment of poly(A) RNA for the ligation of 5′ RNA adaptors but use either enzyme digestion (PARE and degradome sequencing) or sonication (GMUCT) to produce small fragments suitable for sequencing. This methodology has been widely applied to budding yeast, Arabidopsis, rice, maize, grape, soybean and poplar as well as mammals including mice and humans for the identification of miRNA targets or mRNA decay intermediates [13–25].
Current degradome data analysis mainly focuses on the identification of small RNA targets. Several tools such as CleaveLand, SeqTar, and PAREsnip have been developed to fulfill this purpose by pairing sequences flanking uncapped 5′-ends with small RNA sequences [26–28]. The tools have been successfully used to uncover known and new miRNA targets in many organisms. As RNA is constitutively synthesized and subject to bulk or specific degradation, the degradome should represent a complex collection of intermediates produced during RNA maturation or decay. A previous analysis of mouse degradome data revealed miRNA-guide cleavage as well as miRNA-independent events including a group of transcripts sharing a CA-repeat motif within the truncated site . Although degradome data could facilitate the study of RNA degradation beyond the RNA silencing pathways, systematic approaches that dissect degradome data to elucidate mechanisms independent of small RNA regulation have not been established.
In this study, we developed a new pipeline for the analysis of RNA degradome data without a prior assumption of small RNA-guided cleavage to investigate potential mechanisms underlying the formation of uncapped 5′-ends. Our analysis revealed short sequence motifs adjacent to uncapped 5′-ends that were conserved across different degradome libraries and species. Based on sequence similarity and the unique location of these motifs, we have proposed potential routes that may contribute to the complexity and the quality of plant RNA degradome data.
Analysis of motifs associated with predominant uncapped 5′-ends
Position-specific motifs surrounding predominant uncapped 5′-ends
The number of uncapped 5′-ends passing the statistical test was highly variable among the different degradome libraries (Additional file 1: Table S1). This might be explained by the total read number of each library or the degree of RNA integrity for each sequencing sample. The uncapped 5′-ends initiated by known miRNAs represented less than 2% of the total unique ends passing the statistical test which suggests that miRNA-independent mechanisms may contribute significantly to the formation of predominant uncapped 5′-ends (Additional file 1: Table S1).
Motifs identified from the analysis of predominant uncapped 5′-ends in Arabidopsis and rice degradome libraries
The majority of motifs could be recovered from the 3′ UTR which is in contrast to that most plant miRNAs target the CDS (Table 1). For most miRNAs of Arabidopsis and rice, targets of a single miRNA family do not exceed 20 . However, motifs identified in this study were often associated with more than 20 sites among 1000 or fewer uncapped 5′-ends used in MEME analysis. Motif 2 was the most significant example, being found in more than 100 sites among 1000 uncapped 5′-ends in the 3′ UTR for three rice libraries (Table 1). The results of motif analyses thus suggest that mechanisms underlying the formation of uncapped 5′-ends containing these short motifs might play prominent roles in the production of predominant uncapped 5′-ends in addition to miRNA regulation especially in the 3′ UTR.
Although the rice INF939 and SC938 libraries were generated from the same study and have similar read numbers , three motifs were identified in the INF939 library but no motifs were discovered in the SC938 library. During data processing, we noticed that many PARE ends from the SC938 library were terminated with “GC” dinucleotides. Therefore, we calculated the base composition of the last five nucleotides for all unique reads in the SC938 library and compared the result with that of the INF939 and NPBs libraries. We also calculated the base composition of rice cDNA for reference. The pattern of base composition was uniform among the last five nucleotides in the rice NPBs library and comparable to that of rice cDNA (Additional file 2: Figure S2). However, a dramatic distortion in base composition was seen in the last two nucleotides of all unique reads in the rice SC938 library and a mild distortion in the INF939 library. As the SC938 library was produced with the use of Mme I digestion which generates a 2-nt sticky end, selection bias might occur during the 3′-end ligation and thus distort the whole dataset.
We then searched the literature and databases for known motifs similar to the motif sequences we identified to reveal potential routes leading to small regulatory RNA-independent uncapped 5′-ends. Conservation of these motifs in different libraries or species other than Arabidopsis and rice was further examined by MORPH. Five motif groups that showed preferential accumulation of uncapped 5′-ends at the same position in Arabidopsis and rice and matched reported motifs or sequences are presented and discussed below.
Presence of snoRNA 5′-ends in RNA degradome
Mature and functional snoRNAs are 70–200 nt uncapped ncRNAs without a poly(A) tail and theoretically would not be captured by poly(T) beads which are used to enrich poly(A) RNA for deep sequencing. Unexpectedly, snoRNA 5′ termini were mostly and precisely found in Arabidopsis and rice PARE data but not the majority of other rice ncRNA 5′-ends. Variable 5′-ends of snoRNAs were also reported in the mouse degradome study . A possible explanation for these unexpected results is that the snoRNAs detected by deep sequencing of uncapped 5′-ends might be polyadenylated intermediates instead of mature forms. Yeast exosome mutants show accumulation of 3′ extended polyadenylated snoRNAs which may represent intermediates during snoRNA maturation . In contrast to polyadenylation on protein coding RNAs, which is a hallmark of mature transcripts, oligoadenylation on snoRNAs serves as a signal for 3′-to-5′ trimming in the exosome. A previous investigation of the 3′-end of poly(A) RNA in Arabidopsis by direct sequencing detected sequences downstream of snoRNA mature 3′ termini , supporting the existence of 3′ extended polyadenylated snoRNAs in wild-type plants. Since the PARE data used in this study only revealed the first 20 nt of uncapped RNA molecules from the 5′-end, it is not known whether plant snoRNAs captured in the degradome data have unprocessed 3′-ends like the snoRNA intermediates found in yeast exosome mutants. As the accuracy and throughput of sequencing transcripts longer than 200 nt have been much improved, a minor modification of the PARE protocol by replacing Mme I digestion with size fractionation for RNA species ranging 70–200 nt may provide a means to study these uncapped but polyadenylated snoRNAs.
Association of uncapped 5′-ends with the PUF binding site
To rule out the possibility that these truncated transcripts appearing in degradome data were artifacts due to the high-throughput procedure, we selected six Arabidopsis and eight rice genes to validate the uncapped 5′-ends upstream of putative PUF binding sites by performing modified 5′ RACE individually. Although validation was not successful for every selected gene, we could clone 5′-ends located 2–3 nt upstream of putative PUF binding sites for two Arabidopsis genes and two rice genes (Figure 3G and H). The low success rate of modified 5′ RACE might be because the tissues or growth conditions we used were different from previous studies.
PUF proteins have been reported to be involved in mRNA decay through promoting deadenylation and in translational inhibition [42, 43]. A recent study reported that human PUF binding sites are significantly enriched around miRNA target sites in the 3′ UTR and it has been demonstrated that PUF binding can induce RNA structural change that enhances miRNA accessibility in human cell lines [44, 45]. Although PUF binding may enhance RNA decay through the miRNA pathway, many miRNAs in animals do not induce site-specific cleavage but promote deadenylation . Moreover, most plant miRNAs target the CDS but not the 3′ UTR of transcripts and no miRNAs have been found in budding yeast, suggesting that uncapped 5′-ends specifically accumulated 2–3 nt upstream of the PUF binding site are unlikely to be the products of miRNA-guided cleavage. Taken together, PUF binding may result in the production of uncapped 5′-ends through an uncharacterized but common mechanism.
Association of uncapped 5′-ends with a poly(A) signal-like element
Association of uncapped 5′-ends with RNA binding motifs
Although specifically truncated termini are commonly the result of endonucleolytic cleavage, stalling of exoribonuclease trimming can also generate precise termini during RNA maturation. For instance, maturation of snoRNA 5′-ends in the nucleus requires trimming precursors with 5′-to-3′ exoribonucleases . The protein binding to conserved snoRNA motifs delineates mature 5′ termini by preventing exoribonuclease processing. Resembling the proteins associated with snoRNAs, plant pentatricopeptide repeat (PPR) proteins bound to chloroplast RNA termini are thought to impede 5′ and 3′ degradation and thus serve as the determinants of chloroplast RNA maturation [52, 53]. Interestingly, small RNAs overlapping PPR binding sites on chloroplast RNAs have been reported in both monocots and dicots [53, 54]. Similarly, small RNAs were enriched at the snoRNA 5′-end in animals and plants [34, 55]. These small RNAs may represent the footprints of RNA binding proteins. Although the formation of nuclear-encoded mRNA 5′-ends generally does not require exoribonucleotlytic trimming, we suspect that when mRNAs are decapped and subjected to degradation by 5′-to-3′ exoribonucleases, the region occupied by RNA binding proteins may be less accessible to exoribonucleases and thus form a relatively stable and defined terminus. Therefore, our results may imply that RNA degradome data contain the footprints of various RNA binding proteins.
Association of uncapped 5′-ends with a CAGAC motif in the 3′ UTR
Sequencing artifacts resulting from non-specific PCR amplification
Interestingly, the motif analysis of the AxIDT, AxIRP, and AxSRP libraries generated by the degradome sequencing with the use of Mme I digestion also revealed an Mme I-site containing motif (motif 10) at the same position but with minor sequence difference (Table 1). Strong enrichment of uncapped 5′-ends immediately downstream of motif 10 could be also observed on the genome-wide scale (Figure 7E). The minor sequence difference between motifs 9 and 10 could be explained by the different 5′ adaptor primers used in library construction for the PARE protocol and degradaome sequencing. For the GMUCT libraries (Col-0 and ein5) which were constructed through sonication instead of enzyme digestion, Mme I-site containing motifs were not recovered by MEME analysis whereas a distinct motif, motif 11, corresponding to the 3′-end sequence of the 5′ RNA adaptor used in the GMUCT method was found at the same position (Table 1) . The enrichment of uncapped 5′-ends immediately downstream of motif 11 was seen but less evident in the GMUCT libraries on a genome-wide scale (Figure 7F). Unlike the PARE method and degradome sequencing, the 3′ terminus of the GMUCT 5′ adaptor primer was a few nucleotides upstream of the 3′ terminus of the 5′ RNA adaptor which ligates to the uncapped 5′-end. This arrangement could help eliminate the artifact of non-specific PCR amplification during the trimming of 5′ adaptor sequence. In summary, these three upstream motifs suggest that non-specific PCR amplification could occur in genome-wide analysis of uncapped ends regardless of the use of enzyme digestion or sonication. This result raises some concern about the presence of this artifact in public genome-wide data of uncapped 5′-ends.
Deep sequencing of uncapped 5′-ends provides an unprecedented opportunity to investigate transient and stable RNA intermediates produced during RNA processing and RNA turnover at the level of the genome. As RNA silencing represents one of many pathways involved in RNA degradation, bioinformatics analysis from a perspective independent of small RNA-guided cleavage is crucial for detailed understanding of degradome data. The motif analysis performed in this study provides clues about the significant but overlooked RNA population in degradome data. Polyadenylated ncRNAs, potential footprints of RNA binding proteins and artifacts derived from non-specific PCR amplification may all contribute to the complexity of RNA degradome data. These findings increase our understanding of RNA species that can be captured by deep sequencing of uncapped 5′-ends and may lead to alternative applications of degradome data in the study of ncRNA processing and the identification of target sites for RNA binding proteins.
The genes, genomic sequences and degradome datasets used in this study were downloaded from the following public databases. Two Arabidopsis PARE libraries, three Arabidopsis degradome sequencing libraries, two Arabidopsis GMUCT libraries, four rice PARE libraries, one soybean PARE library and one yeast PARE library were retrieved from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) [13–15, 18, 19, 21, 23, 25]. The accession numbers of 13 libraries are listed in Additional file 1: Table S2. For PARE libraries, only 20-nt reads were used in mapping and subsequent analyses while the first 20 nt of reads were used for GMUCT libraries. Reference sequences and the annotation of Arabidopsis and rice genomes used in mapping uncapped reads were downloaded from TAIR (http://www.arabidopsis.org/, TAIR 10) and MSU Rice Genome Annotation (http://rice.plantbiology.msu.edu/, Release 6.1). Rice snoRNAs and putative intermediate-sized ncRNAs were collected from the report of Liu et al. . Known Arabidopsis and rice miRNA targets previously used to evaluate the performance of the SeqTar method were adopted in this study . Yeast genome sequence was downloaded from Saccharomyces Genome Database (http://www.yeastgenome.org/) and the sequences of yeast 3′ UTR were based on the annotation used in the previous yeast PARE study . Soybean genome sequences and annotation were retrieved from phytozome (http://www.phytozome.net/soybean.php).
To discover position-specific motifs associated with predominant uncapped 5′-ends in each genomic region, the standalone MEME suite was used in the analysis of 50-nt sequences (25-nt upstream and 25-nt downstream) flanking selected uncapped 5′-ends with the following parameters: 6–8 nt motifs which occur zero or once in the given strand per input sequence and each motif must occur at least at five sites .
Motif-oriented read positioning heat map (MORPH)
Cluster analysis and heat map graphing were carried out with R statistical software (http://www.r-project.org/) to visualize the distribution of normalized uncapped reads surrounding motifs on a genome-wide scale. The position of an uncapped read was defined by its 5′ terminus relative to the first nucleotide of motifs which was set as 1. Positions upstream of motifs were indicated by negative values while downstream positions were indicated by positive values. Uncapped reads occurring within a 20-nt region flanking every motif site found in a genomic region were extracted. Next, the read number at each position was normalized by the total reads occurring within the 20-nt region for each locus. Finally, loci were clustered based on the distribution of normalized read numbers across the 20-nt region by Ward’s method with R package.
Plant materials and RNA isolation
Rice (Oryza sativa ssp. japonica cv. Tainung 67) was hydroponically cultured in half-strength Kimura B nutrient medium under a 16/8-h light/dark period and 30/28°C day/night temperature. Arabidopsis thaliana (ecotype Col-0) used in this study was grown on 0.8% Bacto-agar plates containing half-strength MS and 1% sucrose under a 16/8-h light/dark cycle at 22°C. Total RNA of 7-day-old Arabidopsis seedlings and 2-week-old rice seedlings were extracted with Plant RNA Purification Reagent (Invitrogen) and MaxTract high-density gel tubes (Qiagen) for the modified 5′ RACE assay.
Modified 5′ RACE assay
Modified 5′ RACE assay was performed to validate uncapped 5′-ends using GeneRacer Kit (Invitrogen). First, poly(A) RNA purified from 50–100 μg total RNA using the MicroPoly(A) Purist Kit (Ambion) was ligated with the 5′ RNA adapter and reversely transcribed with the oligo-dT primer. cDNA was used as template for nested PCR analysis. The primary PCR was performed using the GeneRacer 5′ primer and a gene-specific primer, followed by secondary PCR using the GeneRacer 5′ nested primer with a gene-specific nested primer. Amplified products of expected size were gel purified, cloned into pJET1.2/blunt cloning vector (Thermo) and sequenced. The primers used in this study are listed in Additional file 1: Table S3.
We thank Ms. Miranda Loney in Academia Sinica for English editing of this paper and Dr. Hsien-Da Huang at National Chiao Tung University for helpful discussions. This work was supported by Academia Sinica.
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