Genomic Organization of Zebrafish microRNAs
© Thatcher et al; licensee BioMed Central Ltd. 2008
Received: 07 November 2007
Accepted: 29 May 2008
Published: 29 May 2008
microRNAs (miRNAs) are small (~22 nt) non-coding RNAs that regulate cell movement, specification, and development. Expression of miRNAs is highly regulated, both spatially and temporally. Based on direct cloning, sequence conservation, and predicted secondary structures, a large number of miRNAs have been identified in higher eukaryotic genomes but whether these RNAs are simply a subset of a much larger number of noncoding RNA families is unknown. This is especially true in zebrafish where genome sequencing and annotation is not yet complete.
We analyzed the zebrafish genome to identify the number and location of proven and predicted miRNAs resulting in the identification of 35 new miRNAs. We then grouped all 415 zebrafish miRNAs into families based on seed sequence identity as a means to identify possible functional redundancy. Based on genomic location and expression analysis, we also identified those miRNAs that are likely to be encoded as part of polycistronic transcripts. Lastly, as a resource, we compiled existing zebrafish miRNA expression data and, where possible, listed all experimentally proven mRNA targets.
Current analysis indicates the zebrafish genome encodes 415 miRNAs which can be grouped into 44 families. The largest of these families (the miR-430 family) contains 72 members largely clustered in two main locations along chromosome 4. Thus far, most zebrafish miRNAs exhibit tissue specific patterns of expression.
As the transcriptional landscapes of eukaryotic genomes are defined, it appears that overall transcription is much more prevalent than previously thought, perhaps by as much as 10-fold greater than that needed to generate mRNAs encoding the majority of protein coding genes . Abundant noncoding RNAs, both short and long, have been identified but for the most part their functional significance remains unknown. Among recently discovered small RNAs, the best characterized thus far are microRNAs (miRNAs) [2, 3]. Direct cloning strategies and bioinformatic predictions based on the presence of conserved hairpin structures and sequences have suggested that animal genomes encode hundreds, perhaps thousands, of miRNAs [4–7]. Cell movement, specification, and development are regulated, in part, by miRNAs, consistent with the fact that expression of these RNAs is highly regulated in a tissue and time-specific manner. miRNAs originate from RNA Polymerase II transcripts  requiring processing by the RNase III-like enzyme, Drosha before nuclear export. From the large primary transcripts, Drosha releases hairpins that are ~70 nucleotides long with extensive pairing of approximately 28 base pairs in the stem . Hairpin precursors are exported from the nucleus in a RAN-GTP dependent manner using Exportin 5 [10, 11]. In the cytoplasm, miRNA precursors are further processed by a second RNase III-like enzyme, Dicer, releasing mature miRNA duplexes of ~22 nucleotides [12–14]. Typically, only one strand of the duplex pairs with a target mRNA as part of a larger dynamic ribonucleoprotein complex referred to as the RNA Induced Silencing Complex (RISC). Argonuate proteins are key components of RISCs and are thought to play an important role in whether the target mRNA is subject to translational repression or cleavage followed by degradation .
miRNAs usually pair with sequence elements (miRNA Recognition Elements; MREs) within the 3' UTR of their target mRNAs but there have been limited examples of pairing in the 5' UTR . Since miRNAs usually pair with incomplete complementarity to their targets, bioinformatic approaches to identify targets are limited and functional analysis is required to prove mRNA:miRNA interactions. Because of this challenge, only a small number of targets have been experimentally proven. Further, since each miRNA can target multiple mRNAs and a single mRNA can be targeted by multiple miRNAs, significant work remains to characterize the full range of miRNA function [17, 18].
Zebrafish have proven to be a valuable model system to investigate miRNA function and characterize miRNA:mRNA interactions. Since the creation of active miRNAs requires cleavage by Dicer, zygotic Dicer mutants and maternal zygotic Dicer mutants have helped define the role of miRNAs during development [19, 20]. Zygotic Dicer null mutants live approximately 14 days, because there is sufficient maternal Dicer mRNA deposited into the oocyte . Maternal zygotic Dicer mutants exhibit more severe developmental defects and die after only 7 days . Thus, the regulation of miRNA expression is critical for early zebrafish development. For example, miR-214 is required for proper muscle formation, miR-375 is needed for pancreatic islet development, and the large miR-430 family is needed for deadenylation and clearance of maternal mRNAs at the midblastula transition [21–23].
External fertilization, fast development, and ease of genetic manipulation make zebrafish a powerful system to study vertebrate development and analyze miRNAs. To facilitate this work, microarrays and in situ hybridization experiments have provided a wealth of knowledge regarding temporal and spatial expression of miRNAs during zebrafish development [13, 14, 24, 25]. Small RNA cloning coupled with bioinformatic prediction have enabled the identification of many zebrafish miRNAs but since genome sequencing and annotation is not yet complete, the exact number of miRNAs remains to be determined. Here, we have utilized existing databases and newly available genomic sequence information to identify and catalog all known and predicted zebrafish miRNAs.
Zebrafish miRNAs and Genomic Locations
Newly Identified Zebrafish miRNAs.
Characteristics of Zebrafish miRNAs.
Zebrafish miRNA Transcriptional Units
Polycistronic Zebrafish miRNAs.
Clustered miR-430 family: 57 members
miR-183, miR-96, miR-182*, miR-182
Clustered miR-430 family: 10 members
miR-133a*, miR-133a-1, miR-1-2
miR-92a-1, miR-19b*, miR-19b, miR-20a, miR-19a*, miR-19a, miR-17a-1, miR-17a*
miR-130a-1, miR-301a-1, miR-454a-1
miR-93, miR-19d, miR-25
miR-363, miR-19c, miR-20b, miR-18c
miR-214, miR-199*, miR-199-1
miR-133b, miR-133b*, miR-206-1
miR-217-1, miR-216a-1, miR-216b-1
miR-216b-2, miR-216a-2, miR-217-2
miR-200b, miR-200a, miR-429
miR-23a-2, miR-27c, miR-27c*
Zebrafish miRNA Families.
mir-93, miR-20b, miR-17a, miR-20a
miR-430a, miR-430b, miR-430c, miR-130j, miR-430i
miR-363, miR25, miR-92b, miR-92a
miR-34b, miR-15a*, miR-456
miR-30a, miR-30b, miR-30c, miR-30d, miR-30e
miR-27a, miR-27b, miR-27c, miR-27d, miR-27e
miR-200b, miR-200c, miR-429
miR-19a, miR-19b, miR-19c, miR-19d
miR-18a, miR-18c, miR18b
miR-181, miR-181b, miR-181c
miR-153a, miR-153b, miR-153c
miR-141, miR-200a, miR-132, miR-212
miR-135, miR-135b, miR-135a, miR-729
miR-133a, miR-133b, miR-133c
miR-130b, miR-301a, miR-301b, miR-301c, miR-130a, miR-130c, miR-454a, miR-454b
miR-125c, miR-125b, miR-125a
miR-10d, miR-10b, miR-10c
miR-101b, miR-101a, miR-199*, miR-144
let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7h, let-7i, let-7j
miR-29b, miR-29a, miR-457b, miR-457a, miR-15a, miR-15b, miR-15c, miR-16b, miR-16a, miR-16c
As more and more miRNAs are identified, it has become ever more apparent that understanding global gene regulation requires identifying the targets of every miRNA and the functional consequences of such targeting. Microarrays have been used extensively to determine global miRNA expression patterns. Complementing such analyses with in situ localization of miRNAs greatly facilitates testing of candidate target genes during zebrafish development [20, 24, 25, 31]. Because of imperfect miRNA:mRNA pairing, computer algorithms to identify specific miRNA targets typically produce lists of several hundred candidate genes. Such lengthy lists can be partially refined by integrating spatial and temporal expression data for both miRNAs and their targets. Further, because many miRNAs share identical seed sequences (Table 4), it is important to identify all miRNAs that may target a given mRNA. Here, we have analyzed the existing zebrafish genome to expand the list of miRNAs to 415. We determined the chromosomal location for each miRNA and grouped them into seed sequence families. In addition, we compiled existing expression data and listed validated mRNA targets. Together, Additional File 1 provides an easy to access database that should prove valuable for those interested in understanding the role that miRNAs play in regulating gene expression.
The ease with which gain-of-function and loss-of-function experiments can be conducted in zebrafish makes it an attractive model system to study miRNA function. For loss-of-function experiments, whether in zebrafish, cultured cells, or other model organisms, it is imperative that all members of a given family be effectively knocked down to generate consistent phenotypes. By examining the data in this paper and in Additional File 1, it is possible to quickly determine functional redundancy between one or more miRNAs. Such knowledge will help to design antisense morpholino oligonucleotides when entire miRNA families need to be knocked down [21, 23].
Based on sequence conservation, we identified 35 new zebrafish miRNAs bringing the total number of miRNAs encoded by the zebrafish genome to 415. Bearing in mind that the zebrafish genome is not completely sequenced and annotated, analysis of the existing data suggests that the majority of miRNAs thus far evaluated are encoded as distinct, tissue specific transcripts with an even split between those contained as part of polycistronic transcripts versus those encoded as monocistronic transcripts.
To identify new zebrafish miRNAs, existing miRNA sequences from mouse were retrieved from the miRNA registry  and compared with the zebrafish and fugu genome using BLAST and Ensemble's Zebrafish or Fugu Genome databases [32, 33]. Predicted alignments that contained one or more mismatches within the seed region of the miRNA were discarded whereas no more than 3–4 mismatches were allowed in 3' regions. Resulting sequences were then evaluated for hairpin secondary structures using Vienna RNA Secondary Structure Prediction Program (RNAfold ). For those potential miRNAs exhibiting mature miRNA sequence conservation and predicted hairpin structures, MiRscan  and ClustalW2  were utilized to compare hairpin precursor sequences to each other and to 50 previously described and highly conserved C. elegan miRNAs. Further, predicted zebrafish hairpins were examined to primarily include those located within the same transcriptional unit as their human or mouse counterpart.
Family members were determined strictly by identical seed sequences (2nd–7th nts from the 5' end). Intronic, exonic, and intergenic miRNAs were determined by location among predicted genes within Ensemble. Polycistronic family members were classified as being within 3 kb of another known or predicted miRNA and showing similar or identical expression. Zebrafish miRNA expression data was compiled from previously published sources and is included in Additional File 1[13, 14, 24, 25].
This work was supported by NIH grant GM 075790 to JGP and a training fellowship to EJT (GM62758).
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