Identification of novel homologous microRNA genes in the rhesus macaque genome
© Yue et al; licensee BioMed Central Ltd. 2008
Received: 24 August 2007
Accepted: 10 January 2008
Published: 10 January 2008
MicroRNAs (miRNAs) are about 22 nucleotide (nt) endogenous small RNAs that negatively regulate gene expression. They are a recently described class of regulatory molecules that has biological implications for tumorigenesis, development, metabolism and viral diseases. To date, 533 miRNAs have been identified in human. However, only 71 miRNAs have been reported in rhesus macaque. The rhesus is widely used in medical research because of its genetic and physiological similarity to human. The rhesus shares approximately 93% similarity with human in genome sequences and miRNA genes are evolutionarily conserved. Therefore, we searched the rhesus genome for sequences similar to human miRNA precursor sequences to identify putative rhesus miRNA genes.
In addition to 71 miRNAs previously reported, we identified 383 novel miRNA genes in the rhesus genome. We compared the total 454 miRNAs identified so far in rhesus to human homologs, 173 miRNA genes showed 100% homology in precursor sequences between rhesus and human; The remaining 281 show more than 90%, less than 100% homology in precursor sequences. Some miRNAs in the rhesus genome are present as clusters similar to human, such as miR-371/373, miR-367/302b, miR-17/92, or have multiple copies distributed in the same or different chromosomes. RT-PCR analysis of expression of eight rhesus miRNA genes in rhesus tissues demonstrated tissue-specific regulation of expression.
Identification of miRNA genes in rhesus will provide the resources for analysis of expression profiles in various tissues by creating a rhesus miRNA array, which is currently not available for this species. Investigation of rhesus miRNAs will also expand our understanding of their biological function through miRNA knockout, knockdown or overexpression.
MicroRNAs (miRNAs) are non-coding small endogenous RNAs with a length of about 22 nt that negatively regulate gene expression by degrading mRNA or impeding protein translation . MiRNA genes are hosted in intronic, exonic or intergenic regions of the genome and are transcribed into primary miRNA (pri-miRNA) by polymerase II. The pri-miRNAs are processed into ~70 nt pre-miRNAs with a hairpin structure by a microprocessor complex composed of Drosha and Pasha [2–4]. The pre-miRNAs are then transported into the cytoplasm by exportin-5, where the RNAase III enzyme, Dicer, cleaves pre-miRNA into ~22 nt mature miRNAs, which are recruited into the RNA induced silencing complex (RISC) localized in discrete cytoplasmic foci, P bodies [5–8]. The RISC targets the mRNAs by perfect match to degenerate the target transcripts or binds the 3'UTR through imperfect base-pairing to block protein translation . Recently computational analysis suggests miRNA may also bind 5'UTR . One miRNA may target more than a hundred genes . The discovery of miRNAs has led us to rethink the conventional mechanisms of gene regulation, and current research is focused on understanding how these small molecules function in biological processes.
Experimental evidence reveals that miRNAs play important roles in a variety of diseases, such as cancer, diabetes, viral infection, cardiac diseases, as well as in stem cell biology [11–18]. Some miRNAs are present in the genome as clusters where multiple miRNAs are aligned in the same orientation and transcribed as a polycistronic structure, which may function synchronously and cooperatively. The human miR-17/92 cluster composed of 5 miRNAs (miR-17, 18, 19, 20, 92) was found to be related to tumorigenesis and promoted tumor angiogenesis through targeting the anti-angiogenic thrombospondin-1 (Tsp1) by miR-19 or connective tissue growth factor (CTGF) by miR-18 to down-regulate their functions [19, 20]. The cluster miR371/373, which is highly expressed in human testicular germ cell tumors, has been demonstrated to function as an oncogene and is capable of overcoming Ras-mediated senescence in human primary fibroblasts . In undifferentiated human ES cells, this cluster is also highly expressed and down-regulated upon differentiation, implicating a role in regulating stem cell self-renewal and differentiation .
To date, 533 human miRNAs have been discovered, and the functions of some of them have been experimentally verified . While rhesus is an outstanding model of human physiology, only 71 rhesus miRNAs have been reported and registered in the Wellcome Trust Sanger Institute miRBase [23–25]. The study of miRNA in this species lags far behind the mouse, rat and human as well as invertebrates and plants. There are no reports on miRNA functions in rhesus thus far. Recently, the whole rhesus genome was sequenced through an international collaboration, which provides an opportunity to dissect the genome and identify miRNA genes .
To facilitate the examination of miRNAs in rhesus, we used human pre-miRNA sequences to query the rhesus genomic database at UCSC Genome Bioinformatics [27–29] for homologous rhesus sequences to predict potential miRNA genes. This approach is feasible because miRNA genes are evolutionarily conserved and the human and rhesus genomes share about 93% identity [26, 30].
MiRNA genes in the rhesus genome
MiRNA Genes in Rhesus Genome
MiRNA gene clusters in rhesus genome
Comparison of MiRNA Clusters in Human and Rhesus
Mature MiRNA Sequencesa
ACU CAAAAUGGGG GCG CUUUCC
ACC CAAAAUGGGA GCA CUUUCC
Cluster miR-367/302b genes are expressed in human and mouse ES cells and down-regulated during development into embryoid bodies [11, 18]. We found this cluster shares 100% homology in mature miRNA sequence between rhesus (located on chromosome 5) and human (located on chromosome 4). This cluster is also well conserved in mouse, but not in rat (Figure 1B, Table 2). Clusters miR-17/92 (Figure 1C, Table 2) is highly conserved among human, rhesus, mouse and rat and has been reported to function as oncogenes and promote tumorigenesis . This cluster is hosted on chr13 in human and chrX in rhesus.
Rhesus miRNA gene families
Rhesus MiRNA Gene Families
Positions in Chrb
105917273 – 105917352
120554305 – 120554376
90121100 – 90121173
92701750 – 92701859
68219893 – 68220002
4659068 – 4659177
68995340 – 68995429
CCACCACCG UGUCC GACACC UU
2383348 – 2383458
CCACCACUG UGUCUGACACC UU
CCACCACCG UGUCUGACACC UU
Detection of mature miRNA from rhesus tissues by performing polyA tailing RT-PCR
Based on homology searching of the rhesus genome by querying with human miRNA precursor sequences, we identified 454 miRNA genes, including 383 novel rhesus miRNAs, some of which are arranged in clusters as previously described for human. All of these miRNA genes and clusters are highly conserved between rhesus and human. RT-PCR analyses confirmed expression of rhesus miRNA genes in several tissues. Tissue specific regulation may indicate specialized roles in cell function and or tissue development. Stringent criteria were employed to identify the 454 rhesus miRNAs in this study and some additional miRNAs may yet be identified. While the rhesus genome was recently sequenced, it has not been fully assembled. In addition, rhesus miRNA genes were identified based on human orthologs, which may fail to identify some rhesus specific miRNAs and putative miRNA genes identified in this study contained at least a 16 nt pairing in the stem arm of the hairpin structure of the mature miRNA . We frequently encounter 13 to 15 nt pairings in this core region from the predicted hairpin structure. We didn't consider them as novel miRNA genes in this study, but they could be potential miRNA genes. For example, hsa-miR-484 and bta-miR-484 have 15 nt and 12 nt pairings in stem arm of hairpin structure, respectively. Both hsa-miR-484 and bta-miR-484 have been verified experimentally as miRNA genes in human and bovine, respectively .
While we were writing this manuscript, Zhang et al. reported rhesus miR-506, 507, 508, 509-1, 509-2, 510 and 514 sequences . These sequences are also predicted in the current study, although there were some differences in nomenclature. We employed the systematic annotation previously described for miRNAs .
In the current study we identified 454 rhesus miRNA genes, including 71 that were previously reported. Identification of miRNA genes from rhesus will eventually provide the resources for analysis of expression profiles via microarray. These tools will help identify candidate miRNA genes associated with specific tissues, cells or biological functions.
Human Pre-miRNA sequences were downloaded from Wellcome Trust Sanger Institute miRBase, release 10.0 [22–24] and used to search the rhesus genome at UCSC Genome Bioinformatics [27–29, 38] for homologous sequences. Chromosomal location of putative rhesus miRNAs was determined at UCSC Genome Bioinformatics [27–29, 38]. ClustalW [28, 42, 43] was used for sequences alignment. Precursor sequences were analyzed for secondary structure using MFOLD [44, 45].
PolyA tailing RT-PCR
Primer Sequences for PolyA Tailing RT-PCR
We acknowledge Dr. Ahmi Ben-Yehudah (University of Pittsburgh) for helpful discussions and Dr. Ronald H Plasterk (Utrecht University, The Netherlands) for review of this manuscript prior to publication. The research was supported by the Magee-Womens Research Institute and Foundation (JY, YS and KEO) and National Institutes of Health grants RR18500, AG024992, HD012913, HD008610 (KEO).
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