Lecture 12

microRNAs and Metazoan Complexity by John R. Finnerty

RNA Diversity

Type Length Function mRNA ~500 - 4500 nt encode proteins tRNA ~80 nt translation rRNA 18S mouse (1.9 kb) translation; catalytic component of ribosomes; (large subunit: 28S mouse (4.7 kb) 5S, 5.8S 28S) (small subunit: 18S) miRNA 21-22 nt bind and generally repress complementary mRNAs siRNAs 21-25 nt similar to miRNAs, bind and generally repress mRNAs long ncRNAs >200 nt regulate transcription; many are not polyadenylated; the Xist RNA (17,000 nt long) plays a key role in X-chromosome inactivation snRNAs ~150 nt part of the spliceosome complex which removes introns from pre-mRNAs to make mature mRNAs snoRNAs 60-300 nt located in the nucleolus, these participate in nucleotide modifications of rRNAs, tRNAs, snRNAs, or mRNAs “One of the most interesting challenges facing paleobiologists is explaining the Cambrian explosion, the dramatic appearance of most metazoan animal phyla in the Early Cambrian, and the subsequent stability of these body plans over the ensuing 530 million years.”

The mystery of the Cambrian explosion “First, although chock full of organic forms, the Ediacaran is remarkably reticent with its animal ancestors—besides sponges, only Kimberella has received broad acceptance as a metazoan, possibly a molluscan metazoan.” The mystery of the Cambrian explosion

“And second, the geologic fossil record is a fairly accurate representation of biotic evolution such that both molecular clock analyses and paleoecological considerations agree that mobile macrophagous animals are no older than about the Ediacaran itself.”

Non-BILATERIA 3 phyla / 20,000 described species

Porifera Ctenophora Cnidaria Deuterostomia Ecdysozoa Lophotrochozoa Chordata Arthropoda Annelida Hemichordata Onychophora Mollusca EchinodermataNematoda Platyhelminthes Acoelomorpha Silicispongiae Calcispongia PROTOSTOMIA 525 MYA

~600 MYA BILATERIA 30 phyla / 1.5 million described species “Explosions” or rapid radiations of particular taxa

Mammals, Pollinating Insects, Angiosperms Dinosaurs Many Insects Orders Gymnosperms

Bony fishes

Coelomate metazoa Pre-coelomate metazoa

Two Early Metazoan Fossil Assemblages

Ediacaran Cambrian Key site Ediacara Hills, Burgess Shale, Australia Canadian Rockies Fossil Sandstone Shale bearing (coarse grain) (fine grain) rocks Range of 580-565 MYA 544-525 dates Two Early Metazoan Fossil Assemblages

Ediacaran Cambrian Niches Sessile benthic Sessile benthic organisms occupied organisms (epifaunal) Drifting pelagic organisms Drifting pelagic Directed swimmers organisms Benthic crawlers Burrowing animals (infaunal) Body plans medusoid forms medusoid forms fronds fronds possible bilaterians? worms e.g., Kimberella, which shelled animals some suggest is a mollusc like animal; undulatory swimmers Dickinsonia which some segmented animals suggested is an annelid- animals with paired like animal. appendages

Two Early Metazoan Fossil Assemblages

Ediacaran Cambrian Ancient NON-BILATERIA NON-BILATERIA metazoan Porifera ! Porifera ! lineages represented Ctenophora " Ctenophora ! Cnidaria ? Cnidaria ! BILATERIA BILATERIA Deuterostomia " Deuterostomia ! Lophotrochozoa ? Lophotrochozoa ! Ecdysozoa " Ecdysozoa ! ! Ediacaran faunal assemblage

Crucible of Creation, Simon Conway Morris,1998

Ediacaran Medusoid & Frond

Charnia masoni

Mawsonites spriggi An Ediacaran Annelid?

Dickinsonia

An Enigmatic Ediacaran Spriggina — Frond or benthic crawler?

head shield

holdfast (anchor) Cambrian fossils—the Burgess Shale

Charles Doolittle Walcott Typical Cambrian Preservation

Burgess Shale—Preservation Quality Pennatulaceans (Cnidaria:Anthozoa)

Ediacaran Burgess extant Charniodiscus Thaumaptilon

Cambrian sponge Eiffelia Burgess sponge

Vauxia gracilenta

Burgess polychaete annelid

Burgessochaeta setigera Burgess Onychophoran

Aysheaia

Peripatus

Burgess Chordate

Pikaia

Branchiostoma Burgess Chordate

Ctenorhabdotus Mnemiopsis

Trace Fossils

Lower Cambrian Upper Cambrian Possible Passive Flow Network Spiral-Sinusoidal

McMenamin & McMenamin, 1990 Cambrian faunal assemblage

Crucible of Creation, Simon Conway Morris,1998

Cambrian infaunal assemblage

Crucible of Creation, Simon Conway Morris,1998 Cambrian faunal assemblage

Crucible of Creation, Simon Conway Morris,1998

Ecological Trigger

“The marine ecosystem of the Ediacaran was primarily benthic, with macroscopic organisms largely restricted to the sediment–water interface, whereas the explosion of animals in the Cambrian changed this two-dimensional world into one of three dimensions with macrophagous eumetaozans invading both the infaunal benthos as well as the pelagos. In fact, the origin of these macrophagous mobile metazoans early in the Ediacaran is most likely the trigger of the Cambrian explosion itself.” Genome Hypothesis

“The genome hypothesis suggests that the metazoan genome has changed through time, initially allowing for a relatively broad exploration of metazoan morphospace, but becoming more and more canalized since the Cambrian, which generally precluded the ability to evolve new high-level morphological innovations once phyla evolved.”

Canalization

A concept developed by Waddington. A reduction the degree of phenotypic variation that is caused by either environmental variation or genomic variation.

Waddington’s “epigenetic landscape.” Canalization

A concept developed by Waddington. A reduction the degree of phenotypic variation that is caused by either environmental variation or genomic variation.

Problems with Genome Hypothesis

“....two problems exist when thinking about this genomic hypothesis. First, contrary to expectation, the genomes of protostomes and deuterostomes, the animals that make up the taxonomic bulk of the ‘‘Cambrian explosion,’’ are not only similar in terms of the developmental tool kit (i.e., the types and diversity of components that regulate gene expression), but much of this tool kit is now known to exist in cnidarians and even sponges. Second, when thinking about the subsequent constraints upon phylum-level body plan evolution, if genomic constraints are operational in metazoan macroevolution, then they must have been acquired numerous times independently by each major phylum of animals.” “Temporal Asymmetry of Morphological Innovation”

“morphological variation higher in earlier representatives as compared to later representatives”

“Temporal Asymmetry of Morphological Innovation”

“morphological variation higher in earlier representatives as compared to later representatives”

Early trilobite species (left, order Redlichiida, early Cambrian) exhibit more morphological variation than later species (right, order Phacopida, Ordivician) “Temporal Asymmetry of Morpological Innovation”

“....not only was morphological variation higher in earlier representatives as compared to later representatives, but that protostomes and deuterostomes have indeed acquired numerous and novel genes with each phylum having its own unique repertoire, and that these genes are continually being acquired by animals through geologic time. We hypothesize that these genes, known as microRNAs (miRNAs), serve to both increase complexity and canalization, and thus they might shape, at least in part, the macroevolutionary history of Metazoa.”

MicroRNAs

The first microRNA, lin-4, was discovered in the nematode C. elegans in 1993.

Cell. 1993 Dec 3;75(5):843-54. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Lee RC, Feinbaum RL, Ambros V. Harvard University, Department of Cellular and Developmental Biology, Cambridge, Massachusetts 02138. Abstract lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction. MicroRNAs

The widespread importance of microRNAs was not recognized until the discovery that let-7 was widely conserved in Bilateria.

Nature. 2000 Nov 2;408(6808):86-9. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B, Hayward DC, Ball EE, Degnan B, Müller P, Spring J, Srinivasan A, Fishman M, Finnerty J, Corbo J, Levine M, Leahy P, Davidson E, Ruvkun G. Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA. Abstract Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. 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. Here we have detected let-7 RNAs of approximately 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.

MicroRNAs

Now, ~500 human miRNAs have been identified. The active transcripts are small, 21-22 nucleotides. They are processed from precursor miRNAs with characteristic hairpin structures. The regulation of miRNAs in animals is complicated. an RNAse

an RNAse RISC = RNA Induced Silencing Complex includes Argonaut protein Identification of key proteins Drosha, Dicer, and Argonaut is used as evidence that microRNA pathway is present in an organism.

Binding of RISC complex to specific transcript with complementarity to microRNA results in blocking of translation and or degradation of mRNA. http://www.micrornaworld.com

RNA interference

http://www.youtube.com/watch?v=cK-OGB1_ELE microRNAs & small interfering RNAs

RNA Interference (RNAi) as a tool

“RNA interference (RNAi) has revealed itself to be a powerful tool for biomedical researchers. This image of two C. elegans roundworms makes the gene-silencing technique’s utility clear. In a mutant worm rendered incapable of performing RNAi (left), a green fluorescent protein (GFP)-labeled histone H2B protein is expressed, fluorescing bright green under ultraviolet light. In a wild-type animal (right), the protein is silenced by RNAi, which is imitated simply by feeding the worms bacteria expressing GFP double-stranded RNA.”

Credit: Jessica Vasale/ Laboratory of Craig Mello

http://www.hhmi.org/news/media/Fluorescence_at_Work/9.html The Peterson et al. thesis “We propose that because phenotypic variation decreases through geologic time, because microRNAs (miRNAs) increase genic precision, by turning an imprecise number of mRNA transcripts into a more precise number of protein molecules, and because miRNAs are continuously being added to metazoan genomes through geologic time, miRNAs might be instrumental in the canalization of development.”

miRNA gains in metazoan lineages miRNA reduces transcriptional noise

Consider a transcription factor (TF) that functions by repressing the transcription of a target gene. There is stochastic variation in the amount of the TF. The variation in the amount of TF would create variation in the amount of the target gene. If the TF also activated transcription of a microRNA gene that repressed translation of the same target gene, there would be less variability in the output of the network–i.e., there would be more complete repression of the target gene.

Amount of activate TF per cell transcription TF micro RNA gene repress repress transcription translation miRNA Amount of target gene target gene target gene RNA protein / cell

Does reducing noise facilitate evolution?

There are three requirements for evolution by natural selection: There must be variation. That variation must be heritable. The heritable variation must contribute to differences in reproductive success or “fitness.”

R = h2S

The response to selection (R) = heritability X strength of selection