"Conservation of the Sequence and Temporal Expression of let-7 Heterochronic Regulatory RNA".

AMY E. PASQUINELLI ^*1, BRENDA J. REINHART ^*1, FRANK SLACK ², MARK Q. MARTINDALE ³, MITZI I. KURODA ⁴, BETSY MALLER ², DAVID C. HAYWARD ⁵, ELDON E. BALL ⁵, BERNARD DEGNAN ⁶, PETER MULLER ⁷, JURG SPRING ⁷, ASHOK SRINIVASAN ⁸, MARK FISHMAN ⁸, JOHN FINNERTY ⁹, JOSEPH CORBO ¹⁰, MICHAEL LEVINE ¹⁰, PATRICK LEAHY ¹¹, ERIC DAVIDSON ¹¹and GARY RUVKUN ¹

¹ Department of Molecular Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114, USA
² Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA
³ Kewalo Marine Lab, Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 96813, USA
⁴ Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA
⁵ Research School of Biological Sciences, PO Box 475, Australian National University, Canberra, ACT 2601, Australia
⁶ Department of Zoology and Entomology, University of Queensland, Brisbane, Queensland 4072 Australia
⁷ Institute of Zoology, University of Basel, CH-4051 Basel, Switzerland
⁸ Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
⁹ Department of Biology, Boston University , Boston, Massachusetts 02215, USA
¹⁰ Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
¹¹ Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA

These authors* contributed equally to this work.

Correspondence and requests for materials should be addressed to G.R.

Two small RNAs regulate the timing of Caenorhabditis elegans development [1, 2]. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA [1, 3, 4], and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA [2]. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs [1, 2, 5, 6]. Here we have detected let-7 RNAs of ~21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila , at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

The sequence and function of both the lin-4 and let-7 small RNAs are conserved in the nematode Caenorhabditis briggsae [1, 2]. BLASTN searches reveal one DNA segment from the Drosophila melanogaster genome sequence, three segments from the human genome sequence on chromosomes 9, 11 and 22 bearing exact sequence matches, and two other human segments on chromosomes 9 and 21 with 20/21 matches to the let-7 RNA (Fig. 1a). Database searches did not detect potential lin-4 homologues in any genus except the Caenorhabditae. Similar stem-loop secondary structures are predicted for precursor transcripts of Caenorhabditae, Drosophila and human let-7 RNAs (Fig. 1a). The mature 21-nucleotide (nt) let-7 RNA may be efficiently processed from this putative precursor because only the mature RNA is detected in C. elegans and most other species (see below). A similarly structured larger transcript is also predicted for the lin-4 RNA, and rare transcripts that may correspond to it have been detected in C. elegans [1].

Figure 1.  let-7 gene sequences. (a), Stem-loop structures of C. elegans, D. melanogaster and Homo sapiens let-7 theoretical longer transcripts. The 21-nt let-7 region is shaded. let-7 genomic regions from C. elegans (Z70203), D. melanogaster (AE003659) and H. sapiens chromosome 22 (AL049853). The two human let-7 homologous genes on chromosome 9 are tandemly arranged and separated by 369 base pairs;clustered with the human chromosome 22 let-7 exact match is an 18/21 match to the let-7 RNA. (b), The 3' UTRs of the D. melanogaster (AA3990768) and Danio rerio lin-41 (AI794385) cDNAs contain let-7 complementary sites.

The let-7 RNA regulates late developmental events in C. elegans  by downregulating lin-41 and perhaps other genes that contain sequences complementary to the small RNA in their 3' untranslated regions (UTRs) [2, 6]. lin-41 encodes an RBCC protein that has  Drosophila and vertebrate orthologues [6]. let-7 complementary sites are present in the 3' UTRs of both the Drosophila and zebrafish lin-41 complementary DNAs (Fig. 1b).

let-7 RNA transcripts of  ~21 nt are expressed in human tissues and Drosophila (Fig. 2). As in C. elegans, only RNAs in the ~21-nt range were detected. The expression levels of the human let-7 RNA varied among tissues, indicating possible cell-type regulation of let-7 expression. As let-7 is expressed late in animal development, it may be significant that the lowest level of human let-7 is observed in bone marrow, which consists of a large proportion of immature cells. The let-7 RNA from each human tissue could be the product of any or all of the several human let-7-like genes.

Figure 2. Expression of let-7 RNA in human and Drosophila. (a), Northern blot of total RNA from mixed stage C. elegans (lane 1) and human tissues (lanes 2-13) probed for let-7 RNAand then stripped and re-probed for lin-4 RNA. 5S rRNA serves as a loading control. (b), Northern blot of total RNA from mixed stage C. elegans (lane 1) and D. melanogaster developmental stages (lanes 2-12), probed as in (a). (c), S1 nuclease mapping detects similar transcripts in C. elegans, Drosophila and human total RNA. A 5'-end-labelled antisense strand probe undigested (lane 1) and digested after hybridization to RNA from wild-type C. elegans (lane2), C. elegans let-7(mn112) null mutant (lane 3), human adult lung tissue (lane 4), Drosophila late pupal stage (lane 5) or transfer RNA (lane 6).

Figure 3. Expression of let-7 RNA is developmentally regulated in lophotrochozoansand deuterostomes. (a), Northern blot of total RNA from mixed stage C. elegans (lane 1), Hydroides elegans trocophore larvae (lane 2) and adults (lane 3), Phestilla sibogae veliger larvae (lane 4) and adults (lane5), probed as in Fig. 2a. (b), Northern blot of total RNA from mixed stage C. elegans (lane 1) and D. rerio developmental stages (lanes 2-8), probed as in Fig. 2a. (c), Northern blot of total RNA from mixed stage C. elegans (lane 1) and Strongylocentrotus purpuratus embryonic stages 3-64 hours after fertilization (lanes2-9), pluteus larvae (lane 10) and adult tissues (lanes 11-12), probed as in Fig. 2a. (d), Northern blot of total RNA from adult Acropora millepora (lane 1), adult Nematostella vectensis (lane2), adult Ophlitaspongia tenuis (lane 3) and mixed stage C. elegans (lane 4), probed as in Fig. 2a. We detected a very weak 21-nt signal in a planula stage Acropora millepora but it was probably caused by contamination from a bilaterian predator because these animals are collected from open sea cultures and we could not repeat this detection in an independent planula sample or in embryonic stage A. millepora.

The expression and function of the let-7 RNA in C. elegans begins during the third larval stage, when the gene specifies a transition from late larval to adult cell fates, and continues at all subsequent stages [2]. Expression of Drosophila let-7 is also temporally regulated:  let-7 RNA is absent until the late third instar, just before metamorphosis (Fig. 2b). At the early pupal stage, there is at least a 10-fold increase in let-7 RNA expression that is sustained to adulthood. Thus, temporal regulation of let-7 is similar in Drosophila and C. elegens, which are distantly related members of the ecdysozoan clade [7].

The size and temporal regulation of  let-7 are also conserved in the more distantly related lophotrochozoan clade, members of which do not moult but have larval and adult stages. For example, in two mollusc species and in a polychaete annelid, let-7 RNA is expressed at the adult stage but not at larval stages (Fig. 3a). Thus, let-7 may function at later stages of these species to regulate developmental progression to the adult.

Vertebrates do not develop through larval stages. But in zebrafish expression of let-7 RNA is also temporally regulated: expression commences in the embryo between 24 and 48 hours after fertilization and continues with strong expression at the adult stage (Fig. 3b). Analyses of other deuterostomes shows expression of a 21-nt let-7 RNA in other vertebrates, as well as in urochordate ascidians, and in a hemichordate (Fig. 4). In the echinoderm Strongylocentrotus purpuratus, let-7 may be regulated at the level of precursor RNA processing rather than transcription; a ~100-nt let-7 RNA is detected during embryonic and early larval development, but at the adult stage the 21-nt  let-7 RNA and possible processing intermediates appear (Fig. 3c).

Figure 4. Phylogenetic comparison of let-7 RNA expression. Phylogenetic tree showing species that do (+) and do not (-) express let-7 RNA. Species in which we detect the conserved developmental pattern of let-7 RNA expression (no let-7 RNA in early stages but let-7 expression by adulthood) are indicated by 'Dev.'. The full genus and species names are as follows: Homo sapiens, Mus musculus, Gallus gallus, Xenopus laevis, Daniorerio, Ciona intestinalis, Herdmania curvata, Saccoglossus kowalevskii, Strongylocentrotus purpuratus, Haliotisasinina, Phestilla sibogae, Hydroides elegans, Drosophila melanogaster, Caenorhabditis elegans, Podocoryne carnea, Nematostella vectensis, Acropora millepora, Platygyra daedalea, Ophlitaspongia tenuis and Reneira sp.

We did not detect convincing let-7 RNA in anthozoan and hydrozoan cnidarian species nor in two poriferan species (Figs. 3d and 4). Plant and unicellular organisms also failed to show let-7 RNA expression, consistent with database searches for those species that have been completely sequenced.

Because all three main clades of bilaterian animals express a let-7  RNA that is temporally regulated, but cnidarian, poriferan and all non-animal species that we analysed do not express a detectable let-7 RNA, we propose that the gene evolved after the divergence of diploblastic and bilaterian animals (Fig. 4). However, the lack of detection of  let-7 RNA in the simpler animal and non-animal species might also reflect sequence divergence or loss of the gene in the particular species tested, rather than lack of these genes in a common ancestor.

Although the observation that let-7 homologous RNAs across phylogeny are temporally regulated does not prove that these RNAs actually specify temporal patterning in this broad range of species, the conservation of sequence, of a longer structured precursor, of the 21-nt length, of temporal regulation and of complementary sites in the lin-41 target in two ecdysosoan and one chordate species, are strong evidence of a conserved function. There are candidate mutations that map to the location of the Drosophila let-7 and lin-41 genes which may provide a way to test the function of these genes in another ecdysozoan.

The let-7 RNA is conserved across bilaterian phylogeny but the earlier acting lin-4 RNA is not. Consistent with this, the let-7 target lin-41 is conserved in Drosophila and vertebrates [6], whereas the lin-4 target lin-14 appears to be unique to nematodes [5]. The let-7-regulated late larval transition in C. elegans  may be ancestrally related to late transitions in other species, for example from larval to reproductive forms, whereas the earlier lin-4/lin-14- regulated transition may be a recent invention of the nematode phylum. Alternatively, lin-4 and lin-14 may evolve more quickly.

The 21-nt length of the let-7 RNA is highly conserved, indicating that this size is central to its function. It may be significant that this length is similar to the 21-25-nt RNAs observed during RNA interference (RNAi)-directed down-regulation of target messenger RNAs 8, 9]. However, there are differences between the mechanisms: let-7 is encoded and only the sense strand is expressed, whereas the sense and antisense small RNAs involved in RNAi are processed from exogenously supplied double-stranded RNAs that are hundreds of base pairs long [8-12]. RNAi degrades target mRNA [8-12], whereas the heterochronic lin-4 RNA affects the translation but not the stability of its target mRNAs [5, 13]. Also, mutations in C. elegans genes that confer resistance to RNAi [14] do not have heterochronic phenotypes indicative of a defect in lin-4 or let-7 regulation of target genes (C. Mello, personal communication). One common feature of RNAi and the putative precursors of the heterochronic RNAs is longer regions of duplex RNA, (Fig. 1a), suggesting that similar processing and amplification pathways could generate these 21-nt RNAs.

Two ~21-nt RNAs regulate C. elegans temporal development, and we argue that one of these RNAs is likely to regulate developmental timing in bilaterian animals. We propose that these types of RNAs be called small temporal RNAs (stRNAs). Genome sequence comparisons and expression analyses among bilaterian animals may reveal additional stRNAs that regulate other developmental transitions.

Methods:

Human total RNA samples were purchased from Clontech. Total RNA preparation, northern analysis and S1 nuclease protection assays were performed as described [2]. Oligonucleotides used as northern probes were let-7 5'-AACTATACAACCTACTACCTCACCGGATCC-3' and lin-4 5'-ATAGTACACTCACACTTGAGGTCTCAGGG-3'. 5S rRNA was detected by ethidium bromide staining of the polyacrylamide gels before transfer. Oligonucleotides used for S1 nuclease analyses were 5'-CTATACAACCTACTACCTCACCGGAT-3' (3' end match to C. elegans genomic region), 5'-ACTATACAACCTACTACCTCACCCCA-3' (3' end match to human genomic region) and 5'-ACTATACAACCTACTACCTCAATTTGC-3' (3' match to Drosophila genomic region).

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Acknowledgements. We thank the following people for RNA and tissue samples: T. Heanue, R. Pearse and C. Tabin for chick; J. Gerhart and M. Kirschner for Xenopus and acorn worm; S. Agarwal for Xenopus; N. Stavropoulos for mouse; C. Unabia and K. del Carmen for annelid and mollusc; H. Bode for Hydra; J. Nardone and S. Ferrari for Arabidopsis; P. Sudarsanam for yeast; and D. Selinger for E. coli. The phylogenetic survey in this work was inspired by the NASA Evolution and Development meetings organized by E. Davidson and C. Golden. This work was supported by an NIH grant from NIGMS to G.R. and a grant from the MGH Fund for Medical Discovery to A.E.P. 

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