We describe a catalog of 203 miRNAs from the first small RNA deep-sequencing experiments in T. casteneum
. Tens of high-throughput sequencing experiments have been performed in Drosophila
giving a total number of 171 miRNAs (miRBase, version 15). Our annotation strategy in Tribolium
is intentionally conservative, but we nonetheless conclude that the Tribolium
miRNA complement is at least 15% greater than that of Drosophila
. Around 68% of the Tribolium
miRNAs have detectable homologs in other arthropods, although the proportion of homologs between two insect species is generally below 40% (), suggesting a relatively high rate of turnover during miRNA evolution in insects. We also characterize 47 miRNAs from the Tribolium
catalog that are present in at least one other arthropod, but not in other invertebrates. This arthropod-specific set includes miRNAs that are expressed during early development in Drosophila
such as mir-11, mir-309, mir-14, mir-305, and mir-275 (Aravin et al. 2003
mir-309 paralogs are also present in early developmental stages (). iab-4 is also arthropod specific, and it functions to modulate Hox gene activity during development (Ronshaugen et al. 2005
). Two of the arthropod-specific miRNAs newly detected in this work were also overrepresented in early embryos (mir-3840 and mir-3830 in ).
Despite some existing controversy, the net gain of miRNAs in the drosophilid lineage is currently estimated between 0.3 and 1.0 gain per My (Lu et al. 2008
; Berezikov et al. 2010
). In our Tribolium
data set, we detect 62 miRNAs not present in any other studied species. Assuming an approximate divergence time of 350 My for holometabolous insects (Wiegmann et al. 2009
), the net gain of miRNAs along the Tribolium
branch is roughly 0.18 per My. The primary sources of error on this number are due to our conservative annotation approach (causing the rate of gain to be underestimated) and missed homologs in other species (leading to an overestimate). Nonetheless, our data support a higher overall net rate of miRNA emergence in the Drosophila
lineage than other insects.
Approximately half of Tribolium miRNAs are clustered in the genome (). We show that this clustering is evolutionarily conserved in insects (). The linkage between miRNAs is more conserved between Tribolium and A. mellifera than between Tribolium and Drosophila, suggesting some rearrangement in Drosophila clusters. An illustrative example is the mir-71/mir-2/mir-13 cluster (mir-2 and mir-13 are themselves paralogs). In invertebrates, this cluster is composed of mir-71 and one or more mir-2/mir-13 sequences. In insects, the cluster is highly conserved, with mir-71 and five mir-2/mir-13 elements in tandem. However, in dipterans, mir-71 has been lost, and in Drosophila, the mir-2 family is fragmented into four loci (). We detected sense and antisense transcription in 5 miRNA loci, but only 2 conserve this bidirectional transcription in Drosophila (mir-307 and iab-4). Indeed, no other bidirectional miRNA in any insect conserves this feature in another species. Bidirectional transcription of miRNAs is therefore not a common stable feature.
Shifts in the processing pattern of miRNA precursors lead to changes in their mature sequences. These changes alter the predicted targeting preferences and therefore function of a miRNA. In total, we have shown that one in five miRNAs conserved between Tribolium
have undergone functional shifts. Around 13% of conserved miRNAs between Drosophila
exhibit seed shifting, and we describe arm-switching events in 11% of the orthologous pairs. Arm switching has been previously overlooked but is an important source of evolutionary novelty. Additionally, more than 40% of miRNA loci exhibit 10-fold differences in the proportions of mature sequences processed from 5′ and 3′ arms. In a significant number of cases, it may therefore be misleading to transfer annotation between orthologous miRNAs based exclusively on conservation. It should be noted that the miRNAs exhibiting shifts between Drosophila
have been conserved over ~350 My and are highly expressed. We can therefore confidently assume that these sequences are functional. Current models of miRNA evolution stress the importance of changes in target sites, whereas miRNA function remains highly conserved (Chen and Rajewsky 2007
). However, the relatively high proportion of functional shifts described here also underscores the importance of changes at the miRNA level during the evolution of gene regulatory networks.
has many developmental features conserved from the last common insect ancestor (Tautz et al. 1994
; Marques-Souza et al. 2008
; Shippy et al. 2008
), and it is emerging as an alternate and complementary model for insect biology (Brown et al. 2003
; Richards et al. 2008
; Peel 2009
). Our results clearly suggest that Tribolium
is a good model to study insect miRNA function. First, Tribolium
miRNAs are more likely to be conserved in other insects than Drosophila
miRNAs. In fact, there are at least 18 miRNAs shared with chordates that can be studied in Tribolium
but not in Drosophila
. Second, clustering patterns of miRNAs are better conserved between Tribolium
and honeybee than between Tribolium
. Further investigation of the Tribolium
miRNA complement will increase our knowledge of the evolution of posttranscriptional regulation in animals and, ultimately, help us to understand the origin of extant body plans.