Molecular Evolution of the non-coding Eosinophil Granule Ontogeny Transcript EGOT – Supplement –
Dominic Rose a,h and Peter F. Stadler b,c,d,e,f,g aBioinformatics Group, Department of Computer Science, University of Freiburg, Georges-K¨ohler-Allee 106, D-79110 Freiburg, Germany. bBioinformatics Group, Department of Computer Science, and Interdisciplinary Center for Bioinformatics, University of Leipzig, H¨artelstraße 16-18, D-04107 Leipzig, Germany. cMax Planck Institute for Mathematics in the Sciences, Inselstrasse 22, D-04103 Leipzig, Germany dFraunhofer Institut f¨urZelltherapie und Immunologie – IZI Perlickstraße 1, D-04103 Leipzig, Germany eDepartment of Theoretical Chemistry, University of Vienna, W¨ahringerstraße 17, A-1090 Wien, Austria f Center for non-coding RNA in Technology and Health, University of Copenhagen, Grønneg˚ardsvej 3, DK-1870 Frederiksberg C, Denmark gSanta Fe Institute, 1399 Hyde Park Rd., Santa Fe, NM 87501, USA hCorresponding author: [email protected]
Supplement 25 August 2011 Table 1 Approximate genomic locations of EGO-B orthologs. The coordinates refer to the unspliced genomic regions of EGO-B. Recall that some entries are based on draft assemblies (GeneScaffolds). These genomes contain the EGO-B gene but the respective coordinates are preliminary. In case of assembly problems (e.g. the gene is covered by different scaffolds), no genomic coordinates are given.
Species Assembly Chr. 5’ EGO-B 3’ EGO-B ± size [nt]
Homo sapiens hg19 chr3 4790878 4793274 - 2397 Pan troglodytes panTro2 chr3 4878075 4880473 - 2399 Gorilla gorilla gorGor3 chr3 4902164 4904567 - 2404 Pongo pygmaeus ponAbe2 chr3 65374639 65377039 - 2401 Macaca mulatta rheMac2 chr2 56276017 56278426 + 2410 Papio hamadryas Pham 1.0 Contig1259 Contig623173 26345 28758 + 2413 Callithrix jacchus calJac3 chr15 56602911 56605332 - 2422 Tarsius syrichta tarSyr1 GeneScaffold 4896 162358 164326 - 1969 Microcebus murinus micMur1 - - - - - Otolemur garnettii BUSHBABY1 GeneScaffold 2768 553545 555837 - 2293 Tupaia belangeri tupBel1 scaffold 127316 516 2657 + 2142 Mus musculus mm9 chr6 108404678 108407558 - 2881 Rattus norvegicus rn4 chr4 143936406 143939404 - 2999 Dipodomys ordii dipOrd1 GeneScaffold 6600 155406 158566 - 3160 Cavia porcellus cavPor3 scaffold 16 32052549 32055063 - 2514 Spermophilus tridecemlineatus SQUIRREL GeneScaffold 3331 244508 246869 - 2361 Oryctolagus cuniculus oryCun2 GL018703 3454423 3456525 2102 Tursiops truncatus turTru1 GeneScaffold 1935 210524 212948 - 2425 Bos taurus bosTau4 chr22 22291950 22294301 + 2352 Equus caballus equCab2 chr16 11378820 11381084 + 2265 Felis catus felCat4 A2 55998823 56001116 - 2294 Ailuropoda melanoleuca ailMel1 GL192717.1 421344 423702 - 2359 Canis familiaris canFam2 chr20 15833826 15836114 + 2289 Pteropus vampyrus pteVam1 GeneScaffold 2203 226110 228253 - 2143 Loxodonta africana loxAfr3 chr12 42811986 42814765 - 2780 Procavia capensis proCap1 GeneScaffold 4371 187873 197725 + 9853 Echinops telfairi TENREC GeneScaffold 5028 354037 357269 + 3233 Dasypus novemcinctus dasNov2 GeneScaffold 4264 285144 287278 - 2135 Choloepus hoffmanni choHof1 GeneScaffold 4676 145093 147373 + 2281 Monodelphis domestica monDom5 chr6 236476850 236479951 - 3102 Ornithorhynchus anatinus ornAna1 X1 44628568 44640839 - 12271 Gallus gallus galGal3 chr12 19134732 19138187 - 3455
2 5'exon 3'exon
1.0 muscle 1.0 muscle multiz multiz clustalw clustalw 0.8 0.8 0.6 0.6 0.4 0.4 phastCons score phastCons score 0.2 0.2 0.0 0.0
0 100 200 300 400 500 600 0 200 400 600 800
genomic position, 5'exon genomic position, 3'exon
Fig. 1. Sequence conservation of EGO-B as indicated by the phastCons program. We have com- puted the phastCons scores for three different alignment approaches using the vertebrate model available at the UCSC Genome Browser: (1) our own muscle alignment, (2) the pre-computed 46-way vertebrate multiz alignment from the UCSC browser, and (3) a clustalw alignment based on our set of orthologs. The peaks are fairly similar, only clustalw slightly differs due to a higher alignment error rate resulting from the lack of consistency transformation or similar alignment refinement/improvement steps. Applied phastCons parameters: –transitions 0.01,0.01 –rho 0.4.
length of 5'exon
manual 800 multiz
600
400 nucleotides 200
0 Rat Cat Dog Cow Sloth K.Rat Hyrax Horse Panda Rabbit Chimp Gorilla Tarsier Tenrec Mouse Elefant Human Rhesus Dolphin Squirrel Baboon Chicken Platypus Megabat M.Lemur Armadillo Opossum Bushbaby Marmoset GuineaPig Orangutan TreeShrew
Fig. 2. Differences in aligned sequence data between our approach and pre-computed UCSC multiz alignments. We plot the sequence portion of the 5’exon of EGO-B for different species and align- ment approaches. There is a large overlap but also substantial differences between the two alignment ap- proaches. An obvious drawback of pre-computed alignments is missing data. For example, the UCSC align- ments do not contain the ortholog of panda because the assembly was not ready at the time the alignments were generated. More strikingly, non-human orthologs are only partially included in the UCSC alignments, since it is a reference (human) based approach. Regions that would cause larger gaps in human, such as mouse or rat which exhibit various exonic insertions, are only partially included in the final alignment. However, partial sequences are crucial for any subsequent analysis relying on valid alignments, i.e. RNA secondary structure prediction.
3 external external external internal
2 kb Scale 4771000 4770000 4769000 4768000 4767000 4766000 :chr3 ENCODE Enhancer- and Promoter-Associated Histone Marks HUVEC (H3K27Ac)
HUVEC (H3K4Me1) RefSeq Genes EGOT ENCODE Digital DNaseI Hypersensitivity Clusters DNase Clusters ENCODE UW Digital DNaseI Raw Signal - 1st (in GM12878 cells) GM12878 Raw
ENCODE UW Digital DNaseI Raw Signal - 1st (in K562 cells) K562 Raw 1
ENCODE UW Digital DNaseI Raw Signal - 1st (in HEEpiC cells)
HEEpiC Raw 1
ENCODE UW Digital DNaseI Raw Signal - 1st (in HRE cells)
HRE Raw 1
Fig. 3. EGOT promoter regions. ENCODE data suggest four possible promoter regions for EGOT (marked by the four arrows). Digital DNase1 hypersensitivity clusters indicate three promoter candidate regions upstream of EGOT. On the other hand, histone marks suggest an internal promoter at the 5’exon of EGO-B. The figure contains only exemplary cell lines. However, the depicted signal peaks, especially the the DNase1 hypersensitivity peaks, are consistently present in numerous cell lines.
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Fig. 4. Traces of evolutionary conserved secondary structures. The minimum free energy struc- tures of the six RNAz-predicted regions are at least partially conserved throughout higher eukaryotes. A sequence/structure-based clustering using LocARNA (Will et al., 2007) visualizes the similarities between the predicted structures in more detail. As expected, the structures nearly perfectly cluster into the six groups.
1 kb chr6 108407500 108407000 108406500 108406000 108405500 108405000 HTS data from GSE20370
Mouse EGOT ortholog
Placental Mammal Basewise Conservation by PhyloP
Fig. 5. Non-coding RNA profiling by high throughput sequencing reveals extragenic Pol-II transcription sites at the mouse EGOT ortholog. The deep sequencing data from (De Santa et al., 2010) available in the Gene Expression Omnibus (under GEO accession number GSE20370) confirm tran- scription of the intronic highly conserved element (HCE) and parts of the 3’end of the mouse EGOT ortholog. Although the data do not validate the full mouse ortholog, we benefit twice from the depicted transcribed regions. On the one hand the two independently transcribed regions at the intronic HCE sup- port our findings that the HCE consists of two independent non-coding as well as protein-coding domains. Next, since it was previously postulated that EGOT may act via siRNAs to repress its targets MBP and EDN (Wagner et al., 2007), the signals at the 3’ end on the other hand might in deed indicate small RNAs that are hosted by EGOT.
4 References
De Santa, F., Barozzi, I., Mietton, F., Ghisletti, S., Polletti, S., Tusi, B., Muller, H., Ragoussis, J., Wei, C., and Natoli, G., 2010. A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLoS Biol 8: e1000384. doi: 10.1371/journal.pbio.1000384. Wagner, L., Christensen, C., Dunn, D., Spangrude, G., Georgelas, A., Kelley, L., Esplin, M., Weiss, R., and Gleich, G., 2007. EGO, a novel, noncoding RNA gene, regulates eosinophil granule protein transcript expression. Blood 109: 5191–8. Will, S., Reiche, K., Hofacker, I., Stadler, P., and Backofen, R., 2007. Inferring noncoding RNA families and classes by means of genome-scale structure-based clustering. PLoS Comput Biol 3: e65. doi:10.1371/journal.pcbi.0030065.
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