Mining small RNA structure elements in untranslated regions of human and mouse mRNAs using structure-based alignment
© Khaladkar et al; licensee BioMed Central Ltd. 2008
Received: 18 September 2007
Accepted: 25 April 2008
Published: 25 April 2008
UnTranslated Regions (UTRs) of mRNAs contain regulatory elements for various aspects of mRNA metabolism, such as mRNA localization, translation, and mRNA stability. Several RNA stem-loop structures in UTRs have been experimentally identified, including the histone 3' UTR stem-loop structure (HSL3) and iron response element (IRE). These stem-loop structures are conserved among mammalian orthologs, and exist in a group of genes encoding proteins involved in the same biological pathways. It is not known to what extent RNA structures like these exist in all mammalian UTRs.
In this paper we took a systematic approach, named GLEAN-UTR, to identify small stem-loop RNA structure elements in UTRs that are conserved between human and mouse orthologs and exist in multiple genes with common Gene Ontology terms. This approach resulted in 90 distinct RNA structure groups containing 748 structures, with HSL3 and IRE among the top hits based on conservation of structure.
Our result indicates that there may exist many conserved stem-loop structures in mammalian UTRs that are involved in coordinate post-transcriptional regulation of biological pathways.
RNA cis elements residing in the UnTranslated Regions (UTRs) of mRNAs have been shown to play various roles in post-transcriptional gene regulation, including mRNA localization, translation, and mRNA stability [1–4]. The function of a cis element can be attributable to its primary sequence or structure. For simplicity, they are called sequence elements and structure elements, respectively. Well-known sequence elements include AU-rich elements (ARE), which contain one or several tandem AUUUA sequences and are involved in modulation of mRNA stability [5, 6], and miRNA target sites, which base-pair with their cognate miRNA molecules and are involved in the regulation of translation or mRNA stability [7, 8]. Well-characterized structure elements include Internal Ribosome Entry Site (IRES)  and Iron Response Element (IRE)  in the 5' UTR, Selenocysteine Insertion Sequence (SECIS) , IRE, and histone 3' UTR stem-loop structure (HSL3)  in the 3' UTR. Each element type exists in multiple genes, and thus can be considered as an RNA motif (similar to the concept of protein motif). IRE and HSL3 elements are highly similar to one another within each type; some divergence has been reported for SECIS ; and there is no extensive similarity in primary sequence or secondary structure among IRES elements . These characteristics reflect the ways that the RNA structures function. In addition, various gene-specific structure elements in 5' or 3'UTRs have been shown to play roles in various aspects of RNA metabolism .
Functional RNA sequence elements in the human genome have been heavily studied in recent years, including elements responsible for pre-mRNA splicing, polyadenylation, and miRNA target sites [13–17]. In contrast, RNA structure elements have been investigated to a much lesser extent, partly due to the difficulties in accurately predicting and aligning RNA structures, and assessing false discovery rate (FDR). Recent developments of genome-wide prediction of RNA structures based on aligned genomes [18, 19] or unalignable regions  have resulted in large numbers of conserved RNA structures. On one hand, all methods reported high potential FDR. On the other hand, these results vary from one another in coverage, indicating that there may exist even more structures to be discovered. Here, we took an approach that is not based on genome alignments, dubbed GLEAN-UTR (g rouping by structural distance a nd on tology for RNA elements in UTRs) to uncover conserved RNA structures in UTRs. We focused on small stem-loop structures. We compared folded RNA structures in UTR sequences for orthologous genes by our RNA structure alignment tool RSmatch . Similar orthologous structures were then compared in an all-against-all fashion to derive RNA structure groups. Using cluster analysis and Gene Ontology (GO) information, we identified RNA structures that exist in multiple genes that share common biological pathways. For 10,448 human genes which were analyzed, we obtained 90 RNA structure groups, containing 748 distinct RNA structures in 3' or 5' UTRs from 698 genes. HSL3 and IRE are among the top hits based on conservation of structure. Using a randomized data set, we estimated FDR of 15% for all the structures. About 12% of the structures overlap genomic regions identified by other whole-genome wide studies for RNA structures. This bioinformatics study lays groundwork for future wet lab examination of putative conserved RNA structure elements in human and mouse UTRs.
Mining RNA structure elements in UTRs
We first extracted UTR sequences from NCBI RefSeq sequences. We then used a "slide and fold" method to construct RNA structures in 5' and 3' UTRs (see Methods for detail). With this method, subsequences in UTRs, 100 nucleotides (nt) long or less, were folded according to thermodynamic properties using the Vienna RNA package . Adjacent subsequences were overlapped by 50 nt. This method can derive RNA structures accurately and efficiently for two reasons: (1) Predicting small structures is more accurate and efficient than for large ones; (2) Structures with size less than 50 nt were folded twice as subsequences of two different larger structures, further increasing the chance of getting accurate RNA structures. We also used the setting in the Vienna package that yielded multiple RNA structures with the same minimum energy for a given sequence to further improve the folding accuracy. On the other hand, since we only obtained RNA structures derived from 100 nt subsequences of UTRs, our discovery was limited to small structures, such as short stem-loops. Thus, large RNA structures, such as IRES and SECIS, are not analyzed in this study. This step resulted in 575,410 RNA structures from human UTRs and 445,106 RNA structures from mouse UTRs.
To group similar RNA structures together, we applied hierarchical clustering to the data. First, using pair-wise structure alignment scores, we derived normalized dissimilarity scores to represent distances among the structures (see Methods for detail). We then constructed a hierarchical tree containing all 2,054 structures based on their mutual dissimilarities (Figure 3B and Additional file 2). The hierarchical tree can be "cut" to yield sub trees that represent RNA groups. Figure 3C gives the distribution of the number of structure groups obtained by cutting the tree at every value of normalized dissimilarity score. We selected values at every percentile of this distribution to derive 100 cut heights, i.e. 1st percentile, 2nd percentile, etc. Using these 100 values to cut the tree, we obtained 57, 904 groups of structures, each containing several RNA structures.
Since we were interested in structures that existed in multiple genes involved in the same pathways, we examined the RNA structure groups by their GO information for the biological process category. We applied the hypergeometric test to measure the significance of association between the genes for a structure group and GO terms (see Methods for detail). A structure group was selected for further analysis if the group was significantly associated with a GO term (p-value < 0.05), and there were at least two genes in the group that were annotated with the significant GO term. To measure how member structures in each selected group are similar to one another, we used a measurement called Cohesive Value (CoV), which was the average of all pairwise similarity scores among structures in the same group. Figure 3D shows the distribution of CoVs against group size for all groups. To assess the significance of the CoVs, we randomly selected the same number of structures from 2,054 structures to form groups and calculated their CoV values. For a given group size, we repeated the process 100 times and used the mean value as the expected CoV for groups of the same size. Since the numbers of structures in a group ranged from 4 to 20, we derived expected values for groups with 4–20 structures (Figure 3E). Groups which had a CoV below the expected values were eliminated. After GO analysis and CoV filtering, we obtained 214 structure groups, corresponding to 1,125 distinct structures.
Top 10 structures from the "highly conserved set" based on structure conservation.
Group ID1 (CoV2)
HIST1H1E: histone cluster 1, H1e
HIST1H2BB: histone cluster 1, H2bb
HIST1H1C: histone cluster 1, H1c
HIST1H2BC: histone cluster 1, H2bc
H2AFX: H2A histone family, member X
HIST2H2AA3: histone cluster 2, H2aa3
GO:0006334 (0) nucleosome assembly
GO:0007001 (0) chromosome organization and biogenesis (sensu Eukarya).
TFRC: transferrin receptor (p90, CD71)
SLC40A1: solute carrier family 40 (iron-regulated...
TFRC: transferrin receptor (p90, CD71)
TFRC: transferrin receptor (p90, CD71)
ALAS2: aminolevulinate, delta-, synthase 2 (side...
FTL: ferritin, light polypeptide
GO:0006826 (0) iron ion transport
GO:0006879 (0) iron ion homeostasis
SIPA1L1: signal-induced proliferation-associated 1 like...
CYCS: cytochrome c, somatic, nuclear gene encoding
SLC11A2: solute carrier family 11 (proton-coupled dival...
GPR85: G protein-coupled receptor 85
CXorf41: chromosome X open reading frame 41
GO:0006810 (0.012265) transport
EPHB1: EPH receptor B1
EPHB3: EPH receptor B3
PPP1R3C: protein phosphatase 1, regulatory (inhibitor) subu...
ATOH8: atonal homolog 8 (Drosophila)
GO:0007169 (0.00033) transmembrane receptor protein tyrosine kinase signaling pathway
GO:0007165 (0.031793) signal transduction
GO:0006468 (0.00927) protein amino acid phosphorylation
PTEN: phosphatase and tensin homolog (mutated in multi...
DGAT2: diacylglycerol O-acyltransferase homolog 2 (mou...
MTSS1: metastasis suppressor 1
PADI3: peptidyl arginine deiminase, type III
GO:0045786 (0.00108) negative regulation of cell cycle GO:0007049 (0.009836) cell cycle GO:0006629 (0.001806) lipid metabolism
KITLG: KIT ligand
SUZ12: suppressor of zeste 12 homolog (Drosophila)
SNAP25: synaptosomal-associated protein, 25 kDa
RBBP7: retinoblastoma binding protein 7
GO:0016568 (0.000785) chromatin modification
GO:0008283 (0.002712) cell proliferation
ID4: inhibitor of DNA binding 4, dominant negative ...
TAF11: TAF11 RNA polymerase II, TATA box binding pr...
NOTCH1: Notch homolog 1, translocation-associated (...
RNF12: ring finger protein 12
GO:0016568 (0.000785) chromatin modification
GO:0008283 (0.002712) cell proliferation
WNT7A: wingless-type MMTV integration site family, me...
TIPARP: TCDD-inducible poly(ADP-ribose) polymerase
ARID4B: AT rich interactive domain 4B (RBP1-like), tr...
ICK: intestinal cell (MAK-like) kinase, transcript va...
LRAT: lecithin retinol acyltransferase (phosphatidylc...
RPIB9: Rap2-binding protein 9
GO:0007275 (0.036763) development
MTM1: myotubular myopathy 1
DYRK3: dual-specificity tyrosine-(Y)-phosphorylation r...
AMPH: amphiphysin (Stiff-Man syndrome with breast canc...
CXADR: coxsackie virus and adenovirus receptor
FLJ38628: hypothetical protein FLJ38628
FBLN5: fibulin 5
SGK: serum/glucocorticoid regulated kinase
GLDC: glycine dehydrogenase (decarboxylating; glycine ...
GO:0007155 (0.027609) cell adhesion
AFF2: AF4/FMR2 family, member 2
TOR1B: torsin family 1, member B (torsin B)
BBC3: BCL2 binding component 3
ABHD2: abhydrolase domain containing 2
EBAG9: estrogen receptor binding site associated, antigen, 9...
GO:0006915 (0.011186) apoptosis
HSL3 and IRE are ranked among the top hits with respect to CoV values (1st and 2nd) as can be seen in Table 1. This result not only validated our approach, but also indicated that other groups of RNA structures may also exist, though probably not as well conserved as HSL3 or IRE. Using the multiple alignment function of RSmatch, we generated a consensus structure for each structure group. In a sense, each structure group represents a putative RNA structure element type. The sizes of the consensus structures ranged from 15 to 31. All groups and structures can be downloaded from our lab web site as a batch file , or searched, retrieved and viewed through an on-line database named GLEAN-UTR DB .
To assess the FDR for our method, we repeated all steps using randomized human and mouse UTR sequences maintaining overall dimer frequencies, and calculated the number of selected entries at each step (Additional file 3). At the last step, this randomized set resulted in 17 groups consisting of 110 human structures. Thus, the FDR is ~18.89% for the groups and ~14.71% for the structures. Of these groups, 3 groups with 14 structures also passed the cross-validation with mouse orthologs, giving FDR ~8.82% for the groups and ~5.96% for the structures.
Comparison with other genome-wide RNA structure studies
We have designed a systematic approach to identify RNA structure elements conserved in human and mouse UTRs which may function coordinately in post-transcriptional regulation of biological pathways. This approach contains three major steps: (1) compare RNA structures between orthologous genes; (2) compare RNA structures among all genes; and (3) select RNA structure groups significantly associated with certain GO terms. Presumably, mRNAs containing RNA structure elements from the same group can be coordinately regulated via trans-acting protein factors, like those having HSL3 and IRE, leading to concerted modulation of a biological pathway. We applied this method to mining small RNA structures in this study, primarily because those structures can be more accurately predicted by RNA prediction programs using only thermodynamic parameters. As more powerful RNA structure prediction programs become available, particularly those reliant on phylogenetic information for structure prediction, this approach can be extended to larger RNA structures. The major strength of our approach is the ability to assign functions to candidate RNA structures in the genome. In addition, it may help improve the accuracy in RNA structure identification, as structures shared by multiple genes can be more reliable than those encoded by a single gene.
The assessment of FDR is critical in RNA structure analysis . Using randomized sequences, we estimated a FDR of 15% for the structures identified in this study. False negative rate or sensitivity is another important issue, particularly in this study in which stringent cutoff values were applied at multiple steps. However, it is difficult to address due to lack of knowledge on true positive structure groups. We examined two well-known RNA structure elements, HSL3 and IRE, for sensitivity. For HSL3 and IRE genes that have orthologous gene information, we found that 35% (6 out of 17) HSL3 elements and 60% (6 out of 9) IRE elements are included in our final result. Thus the sensitivity can be low for some structure groups and high for others. Several steps can result in exclusion of conserved functional RNA structures in our method. First, the current coverage of orthologous genes and UTRs is not complete. In fact, most of the human HSL3 true positive structures (44 in total) were not even analyzed in this study due to lack of orthologous gene or UTR information. This will improve as more comprehensive gene annotations, and more accurate transcription start sites and polyadenylation sites are available. Second, it is known that RNA structure prediction by thermodynamic parameters has limitation in accuracy . Third, some structures may reside in genes for which GO information is not adequately annotated.
One potential approach to improve sensitivity is to search the genome with consensus RNA structures derived from the groups. We tested this idea by first generating RNA structure patterns for the groups and used them to search human UTRs by PatSearch . Candidate elements were further analyzed for GO terms to ensure consistency in their association with biological pathways as the original groups. As expected, the group size increased exponentially (Additional file 5). While this approach seems promising in reducing the false negative rate, the control for false positive rate needs to be further developed. We leave this work for future exploration.
About 12% of the structures identified in this work overlap those reported in other studies (Figure 4). Interestingly, each genome-wide approach resulted in a large fraction of unique structures, suggesting that RNA structure identification is largely influenced by the chosen method. Many structures in UTRs identified by other studies are not in our final result (Figure 4). This is attributable to several aspects of the design of our study, in addition to the technical difference and false negative issues described above. First, our analysis is based on RNA structure groups, and functional structures located in individual genes are not included. We found this is the case for several recently reported RNA structures in UTRs [28, 29]. Second, RNA structures with similar functions but different secondary structures, like IRES, cannot be identified. Third, large structures, like SECIS, are not examined. Notwithstanding these issues, the structures that overlap between this study and others are of higher importance for further wet lab validations (Additional file 4).
In summary, our result indicates that there may exist many conserved stem-loop structures in human UTRs that are involved in coordinate post-transcriptional gene regulation of biological pathways, similar to HSL3 and IRE structures. This bioinformatics study lays a ground work for future wet lab validations of putative RNA stem-loop groups and represents a framework which can be used to analyze RNA structures identified by other approaches and in other species.
UTR sequence and structure databases
We downloaded 28,926 human and 26,243 mouse RefSeq mRNA sequences from NCBI. UTRs of RefSeq sequences were extracted according to RefSeq's GenBank annotation. The information regarding human and mouse orthologs was obtained from the HomoloGene database. We prepared RNA structures in UTRs by a method called "slide and fold" as described previously . Briefly, for each UTR sequence, we took 100 nt subsequences at every 50 nt nucleotide position from 5' to 3' resulting in consecutive subsequences overlapping with one another on a 50 nt segment. Subsequences shorter than 100 nt, e.g. at the 5' or 3' ends, were also kept. We then folded all of the subsequences using the RNAsubopt function in the Vienna RNA package , with the setting "-e 0". With this setting, multiple structures with the same minimum energy can be generated. Using this method, we obtained 575,410 structures from human UTRs, and 445,106 structures from mouse UTRs.
RNA structure comparison
Pairwise comparisons of RNA structures (human vs. mouse and human vs. human) were carried out by RSmatch , with the "dsearch" function and default scoring matrices for ss and ds regions. Specifically, nucleotide match scores were 1 and 3 in single-stranded (ss) and double-stranded (ds) regions, respectively; and mismatch scores were -1 and 1, in ss and ds regions, respectively. Gap penalty was -6 for both ss and ds regions. This scoring scheme in effect gave more weight on matches in ds regions than those in ss regions. Randomization of RNA structure was carried out by a PERL script.
Cluster analysis of RNA structures
To cluster RNA structures, we calculated normalized dissimilarity scores Di, j between all structures: Di, j = (Smax-Si,j)/Smax, where Si, j was the similarity score derived from RSmatch using the local structure alignment function between structures i and j, and Smax was the maximum similarity score obtained from all structure comparisons. For cluster analysis, we used the hierarchical clustering function in R , with the "average linkage" method for joining nodes. To select groups of RNA structures, we applied the "cutree" function to cut the hierarchical tree obtained from R into groups using the normalized dissimilarity scores, which were also called heights in the tree. Structures in each group were aligned by the multiple structure alignment function of RSmatch with default scoring matrices. Structures in the same group were also compared in a pairwise manner; the average of all pair-wise similarity scores for the group was called the Cohesive Value (CoV) of that group, which indicated the degree of similarity among structures in the group.
Gene Ontology analysis
For each RNA structure group M containing multiple genes, all GO entries are examined to evaluate their associations with M . Through the mapping information between M and a GO entry G in a GO category C, we are able to calculate four numbers: (1) N1, the number of genes associated with any GO entry in C, (2) N2, the number of genes associated with G in C, (3) N3, the number of genes in M associated with any GO entry in C, and (4) N4, the number of genes in M associated with G in C, where N1 ≥ N2 and N3 ≥ N4. The p-value of the GO entry G is calculated by p(G) = f(N4, N2, N1 – N2, N3), where the function f is defined in equation 1.
Cross-validation with mouse UTR structures
After performing the GO analysis and CoV filtering, we cross-validated selected human RNA structure groups with their orthologous mouse structures. For each group, mouse UTR structures corresponding to human structures in the group were retrieved. Then the mouse UTR structure which has the highest similarity to a human structure was selected. All these selected mouse structures were compared by the multiple structure alignment function of RSmatch which also gave the consensus structure. The consensus structure of human RNA structures was then compared to that of mouse one. An RNA structure group was considered to be highly conserved if: (1) the human consensus was identical to the mouse one, or (2) the human consensus was contained within the mouse one or vice verse. In case (2), a consensus of human and mouse structures was built to represent the structure group.
Comparison with structure elements from other studies
The datasets for Pedersen et al. and for Washietl et al. were downloaded from their respective web sites [18, 19]. The dataset from Torarinsson et al. was obtained from the authors. We used BLAT to find genomic locations for all structure elements, including ours, and identified overlapped ones by their locations.
We thank Elfar Torarinsson for sharing results, and members of BT lab for helpful discussions. This work was partially supported by a grant from The Foundation of University of Medicine and Dentistry of New Jersey to BT.
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