1 ENCODE Pseudogene Summary for GT call Mark Gerstein 2005, :00 EDT summary of 6 Calls: Sept. 15, 22; Oct. 6, 13, 20, 27

2 Developed Consensus Set of 198 Pseudogenes ADerived from a qualified union of GIS, Havana, UCSC, & Yale with a uniform criteria on boundaries 1.Identify a “good” set of human proteins – HAVANA set? 2.Remove pseudogenes (from all 4 groups) overlapping with current GENCODE exons (does GENCODE have an updated version?). 3.Create an union of the remaining pseudogenes. 4.Find the “best” matching proteins for each pseudogene, remove entries without a BLAST hit (e-value cutoff issue?). 5. Realign each pseudogene to its parent protein to produce a uniform alignment and to define the start and end coordinates. 6.Apply a threshold to sequence identity and coverage? (No.) 7.Classify pseudogenes into processed and non-processed (how?) BOverall 222 pseudogenes; application of above receipe gives 198 Consensus (Intersection set of above is 81 (proc) + 49 (non-proc)) CCurrently, on test browser + encode wiki From Deyou Z. + Robert B.

3 Interesting Complexities of Pseudogene Annotation: Insertion of One Pseudogene into Another One heterogeneous nuclear ribonucleoprotein A1 (HNRPA1) pseudogene (parent on Chr12) NADH dehydrogenase 2 (MTND2) pseudogene (parent mitochondrial) NADH dehydrogenase 4 (MTND4) pseudogene (parent mitochondrial) cytochrome b (CYTB) pseudogene (parent mitochondrial) First insertion event Remnant of a second, mitochondrial insertion event (has post-insertion deletions) Protein evidence From Adam F.

4 Frameshift LILRA3 LILR pseudogene EST Evidence of Expression from a Pseudogene at 5’ UTR of Known Gene Upstream pseudogene corresponds to exons 1-3 of LILR family genes, 3’ exons have been lost. EST evidence supports expression from the pseudogene locus extending to known gene LILRA3. From Adam F.

5 - # of 198 overlapped by interrogated regions (affy arrays): 180 (90.9%) - # of 198 overlapped by yale tars or affy transfrags (union): 106 (53.5% of all ; 58.9% of interrogated) => There is evidence of transcription (from tars or transfrags) of the pseudogene or the parent gene (if cross-hybridization) for 53.5% of the consensus pseudogenes (upper bound on transcription) - # overlapping cage tags: 11 (5.5%) - # overlapping ditag tags: 1 (0.5%) (83 (41.9%) are overlapped by full length ditags) From France D. TAR/Transfrag Evidence for Transcription in 198 consensus pseudogenes

6 Example Pseudogene overlapped by tars/transfrags and tags: ENCODE_consensus_187 but pseudogene is 93% similar to parent From France D.

7 Consensus Pseudogenes with ≥2 ChIP-chip Hits Pgene-IDPgene-typeE2FH3K4me3 (0h & 30h) Sp3STAT1 13Processed Processed Processed Processed Processed Processed Non-Processed0100 [ 177 ]Non-Processed Processed Processed0011 Has Trans- criptional Evidence (intersects Gencode transcript) From Deyou Z. Look for ChIP-chip hits upstream of the pseudogenes

8 From Deyou Z. Pot. Transcribed Pseudogene (#177) with Upstream ChIP-chip Hits

9 Experiments to Validate Expression of Encode Pseudogenes Select ENCODE pseudogenes from the intersection part of consensus set –49 non-processed, 125 processed Designed oligos (25mer, Tm 70°C) –Either specific to pseudogene or shared between parental gene and pseudogene Doing 5’RACE in 12 human tissues –Brain, heart, kidney, spleen, liver, colon, sm. intestine, muscle, lung, stomach, testis, placenta –First 96 pseudogenes 5’RACEs done in 12 tissues –Last 78 will be done next week To do: pool multiple RACEs, send to Santa Clara and hybridize to Affymetrix ENCODE 20 nucleotide resolution arrays From Alex R. Stylianos Antonarakis, Robert Baertsch, Jorg Drenkow, Tom Gingeras, Charlotte Henrichsen Philipp Kapranov, Catherine Ucla, Alexandre Reymond Affymetrix, UCSC, University of Geneva, University of Lausanne

10 Extra Slides

11 Pseudogene group Core people: Jennifer Harrow,WEI Chia-Lin,Adam Frankish, "Dike, Sujit",Robert Baertsch, imim.es,Deyou Zheng, Yontao Lu unige.ch, Others: "Hoyem, Tara L", Roderic Guigo Serra, "'Gingeras, Tom'“ Suganthi Balasubramanian 6 Calls : Sept. 15, 22; Oct. 6, 13, 20, 27

12 81 (34) Havana-Gencode: 165 pseudogenes ( ) Yale: 167 pseudogenes ( ) UCSC retrogenes: 146 not expressed 16 (7) 33 (1) 15 (1) 17 (2) 16 (0) 54 (2) Refresher: many repetitions of the below “Venn analysis” 7 Havana agrees to be added (8, 11, 40, 59, 139, 152, 169). 4 at coding loci. [Yale agrees to delete] 1 with weak sequence identity.* 5 with “non-real” proteins.* 9 Havana agrees to be added. 2 at coding loci. [Yale agrees to delete] 1 with weak sequence identity.* 2 with “non-real” proteins.* * Solved by consistent protein set & threshold Numbers according to Adam’s note

13 Rearranged exon order in unprocessed pseudogene Protein evidence adaptor-related protein complex 1, beta 1 subunit (AP1B1) pseudogenes Exon 6Exon 3 Splice sites same as parent gene Dot plot protein evidence vs genome Following duplication of the AP1B1 locus rearrangements/duplications have produced two unprocessed pseudogenes corresponding to exons 6 and 3 of the parent gene From Adam F.

14 Rearrangement of processed pseudogene pseudogene similar to part of ribosomal protein L3 (RPL3) Protein dot plot mRNA dot plot Following insertion, one end of the RPL3 pseudogene has been flipped onto the opposite strand (with some loss of internal sequence) From Adam F.

15 Overlaps by tar/transfrag subset - Nb overlapped by interrogated regions (affy arrays): 180 (90.9%) - Nb overlapped by yale tars or affy transfrags (union): 106 (53.5% of all ; 58.9% of interrogated) - Nb overlapped by yale tars (union): 84 (42.4% of all ; 46.7% of interrogated) - Nb overlapped by affy transfrags (union): 102 (51.5% of all ; 56.7% of interrogated) - Nb overlapped by polyA+ tars/transfrags (union) 105 (53% of all ; 58.3% of interrogated) - Nb overlapped by total RNA tars (union) 61 (30.8% of all ; 33.9% of interrogated) From France D.

16 Expression from pseudogene locus (1) – putative novel transcript HAVANA sialyltransferase pseudogene (RP3-477O4.5) supported by protein evidence Putative novel transcript supported by a single EST with has a polyA site and signal Supporting EST (100% ID) Aligned proteins (column collapsed) polyA site and signal Appears to be some transcription from this locus which is supported at the 3’ end by a single EST From Adam F.

17 Intersect Consensus Pseudogenes with ChIP-chip Hits FactorsE2FH3K4me3 (0h) H3K4me3 (30h) Sp3STAT1 GroupUCDavisUCSD StanfordYale Total Hits Known Genes (405)  genes (198) From Deyou Z.