500 1000 1500 Four ribosomal

Y37E3.8 Y37E3.7 protein -containing rpl-27a rpa-1 operons from 500 1000 1500 2000 Caenorhabditis elegans

F25H2.10 F25H2.11 rpa-0 tph-1 500 1000 1500 2000 2500

T01C3.7 T01C3.6 fib-1 rps-16 500 1000 1500 2000 2500

F37C12.9 F37C12.4 F37C12.3 rps-14 rpl-36 ayc-1 500 1000 1500 Conserved operons

Y37E3.8 Y37E3.7 identified in rpl-27a rpa-1 Brugia malayi 500 1000 1500 2000 Oscheius tipulae Pristionchus pacificus F25H2.10 F25H2.11 rpa-0 tph-1 Strongyloides ratti 500 1000 1500 2000 2500 Ascaris suum

T01C3.7 T01C3.6 fib-1 rps-16 500 1000 1500 2000 2500

new in some taxa F37C12.9 F37C12.4 F37C12.3 rps-14 rpl-36 ayc-1 intron absent in some taxa Operon sizes in sequenced genomes

10000

1000

MI 100 BM CE

Number of operons

10

1 2 3 4 5 6 7 8 9 10 Size of operon ()

B. malayi C. elegans Number of M. incognita genes with 14503 8098 orthologues Number of genes with orthologues in M. 3083 (21%) 1879 (23%) incognita operons Number of M. incognita operonic genes 336 516 with orthologues also in operons Number of operons conserved with M. 3 9 incognita

intron exon intron exon intron exon x

transcripton ATG STOP POLYA start 5’ UTR 3’ UTR

exon intron exon intron exon intron exon pre-mRNA x transcripton ATG gt ag gt ag gt ag STOP POLYA start 5’ UTR 3’ UTR

exon exon exon exon mRNA CAP AAAAAAAAAAAAAAAAA ATG STOP

5’ UTR 3’ UTR cis-splicing of exon intron exon intron exon intron exon x

transcripton ATG STOP POLYA start 5’ UTR 3’ UTR

outron exon intron exon intron exon intron exon pre-mRNA x tttcag ATG gt gt ag gt ag STOP POLYA

5’ UTR 3’ UTR donatron SL1 *CAP gt

exon exon exon exon mRNA *CAP SL1 AAAAAAAAAAAAAAAAA ATG STOP

5’ UTR open reading frame 3’ UTR trans-splicing of leader sequence gene 1 gene 2 x

transcripton start

gene 1 gene 2 polycistronic x pre-mRNA tttcag

*CAP SL1 *CAP SL2

mRNA(gene1) *CAP SL1 AAAAAAAAAAAAAAAAA mRNA(gene2) *CAP SL2 AAAAAAAAAAAAAAAAA trans-splicing of operonic genes A L S L Brugia malayi OP1032 248 bp behaves like an operon BUT the downstream gene B + - M is trans-spliced rpl-27a rpa-1 3054 2036 to SL1 1 1636

2 1018 3

506 gene 1 gene 2 x

gene 1 gene 2

C. elegans *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL2-like AAAAAAAAAAAAAAAAA

C. briggsae *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL2-like AAAAAAAAAAAAAAAAA

B. malayi *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL1 AAAAAAAAAAAAAAAAA gene 1 gene 2 x

gene 1 gene 2

C. elegans *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL2-like AAAAAAAAAAAAAAAAA

C. briggsae *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL2-like AAAAAAAAAAAAAAAAA

N. brasiliensis *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL2-like AAAAAAAAAAAAAAAAA

P. pacificus *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL2-like AAAAAAAAAAAAAAAAA

S. ratti *CAP SL1-like AAAAAAAAAAAAAAAAA *CAP SL1-like AAAAAAAAAAAAAAAAA

A. suum *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL1 AAAAAAAAAAAAAAAAA

B. malayi *CAP SL1 AAAAAAAAAAAAAAAAA *CAP SL1 AAAAAAAAAAAAAAAAA SL2 The nematode Strongylida Rhabditida operonic transcription V Diplogasterida & trans-splicing variant SL1 Strongyloididae system appears IVa Steinernematidae to have been operons Panagrolaimidae assembled piecemeal ??? Cephalobidae Aphelenchida through IVb Tylenchida evolutionary time. SL1 Oxyurida Spirurida III Ascaridida Rhigonematida C Chromadorida Enoplida II Triplonchida Dorylaimida The C. elegans system Mermithida (SL2-like spliced leaders) I Trichocephalida Mononchida is highly derived. Outgroups Mark Blaxter – Comparative Nematode Genomics - 2008

Trichinella spiralis trans-spliced leaders Sr_SL1s GGTTT—-AAAAAACCCA-GTT----TAAG Ts_SL14 GAATT--AAAAAGAAGACGCT----GAAG Ce_SL2 GGTTT-----TAACCCA-GTT-ACTCAAG Ts_SL11 AACCTTT-----GAACCCA-CTT----CAAG Ts_SL1 CAGGTATTT------ACCAG-ATCTA--AAAG TS_SL2 AGGTATTT------ACCGA-ATTAA--AAAG Ts_SL3 GGTTATTT------ACCGA-ACTTA--AAAG Ts_SL4 GCGAATTG------TTCGAATTTACTTGAAG Ts_SL5 AAATACCTTT-----CAATTTG-TTT----GAAG Ts_SL6 AACCTTT-----GCGCATC-GTTT---AAAG Ts_SL7 AACCTGC-----ACGACTT-GTTC---GAAG Ts_SL8 ATCTG------TCGGT-ATTCCTGAAAG Ts_SL9 AGACG------TGGTT-ATTTATTGAAG Ts_SL10 GGTAATATTT------ACTGA-ATT----CAAG Ts_SL12 ACGAATTT------ACCGT-ATTTGT-CAAG Ts_SL13 TACCATT-----CAATTTA-TTTT---GAAG Ts_SLZ GCTA-----TGACCATGATTACGCCAAG Ts_SLY T-----TGACACTGATTACGCCAAG Ts_SLX AGC-----TTGACCTGATTACGCCAAG

Pettitt et al. 2008 RNA 14:760-770 SL2 The nematode Strongylida Rhabditida operonic transcription V Diplogasterida & trans-splicing variant SL1 Strongyloididae system appears IVa Steinernematidae to have been operons Panagrolaimidae assembled piecemeal ??? Cephalobidae Aphelenchida through IVb Tylenchida evolutionary time. SL1 Oxyurida Spirurida While SL1 is a III Ascaridida synapomorphy for Rhigonematida Rhabditida, other facets tSL Chromadorida C of the system Enoplida have limited distribution. II Triplonchida Dorylaimida The C. elegans system Mermithida (SL2-like spliced leaders) I Trichocephalida variant SLs Mononchida is highly derived. Outgroups EXON

INTRON

SPLICED LEADER

EXON +

INTRON trans-splicing

pre-mRNA SL RNA mRNA

EXON +

A

A

A

A

A

A

INTRON A

EXON

A

A A

A

A A SL trans-splicing (Philodina spp.) Platyhelminthes ATG

Cnidaria

Trans-spliced leader addition to mRNAs in a cnidarian. Stover N & Steele RE. Proc Natl Acad Sci U S A. 2001 98:5693-8

Surprising diversity and distribution of spliced leader RNAs in . Davis RE. Mol Biochem Parasitol 1997 90:395-7. Oikopleura dioica Ascidians (sea squirts)

Ciona intestinalis

ATTCTATTTGAATAAG TTCTATTTGAATAAG TCTATTTGAATAAG CTATTTGAATAAG ATTTCTATTTGAATAAG AATTCTATTTGAATAAG GTATTCTATTTGAATAAG ATTCTAATTGAATAAG ATTCTATTAGAATAAG ATTCTA-TTGAATAAG ATTCTATTTCAATAAG ATTCTATTTGAAAAAG ATTCTATTTGAACAAG ATTCTATTTGAAGAAG ATTCTATTTAAATAAG ATTCTATTTGA----- ATTCTGTTTGAATAAG

Genomic overview of mRNA 5'-leader trans-splicing in the ascidian Ciona intestinalis. Satou Y, et al., Nucleic Acids Res. 2006 34:3378-88.

Spliced-leader RNA trans splicing in a chordate, Oikopleura dioica, with a compact genome. Ganot P, et al., Mol Cell Biol. 2004 24:7795-805. Choanoflagellida Porifera Placozoa Metazoa Acoelomorpha Rhombozoa Orthonectida Multiple, Myxozoa Mollusca Echiura Pogonophora independent Annelida Entoprocta Brachiopoda Bryozoa events of Sipuncula L Rotifera Acanthocephala Platyhelminthes gain of Cycliophora Gnathostomulida Nemertea SL trans- ? Gastrotricha Micrognathozoa Tardigrada E Arthropoda splicing Onychophora Priapulida Kinorhyncha Loricifera and operons D Nematomorpha ? Nematoda Xenoturbellida Echinodermata Hemichordata Chordata Choanoflagellida Porifera Placozoa Cnidaria Metazoa Ctenophora Acoelomorpha Rhombozoa Orthonectida Multiple, Myxozoa Mollusca Echiura Pogonophora independent Annelida Entoprocta Brachiopoda Bryozoa events of Sipuncula L Rotifera Acanthocephala Platyhelminthes loss of Cycliophora Gnathostomulida Nemertea SL trans- Gastrotricha ? Micrognathozoa Tardigrada E Arthropoda splicing Onychophora Priapulida Kinorhyncha Loricifera and operons D Nematomorpha ? Nematoda Chaetognatha Xenoturbellida Echinodermata Hemichordata Chordata Choanoflagellida Porifera Placozoa Cnidaria Ctenophora Acoelomorpha Rhombozoa Orthonectida Myxozoa Nematoda Nematomorpha Loricifera Kinorhyncha Priapulida Onychophora Arthropoda Tardigrada Micrognathozoa E Gastrotricha E Nemertea Gnathostomulida Cycliophora Platyhelminthes Acanthocephala Rotifera Sipuncula L Bryozoa L Brachiopoda Entoprocta Annelida Pogonophora Echiura D Mollusca D Chaetognatha Xenoturbellida Echinodermata Hemichordata Chordata