Titin exon specific GERP scores

Coming back to Titin gene, are all exons equally important? GERP scores could tell us something about how the exons vary in conservation level at base-pair resolution.

In the above figure, the y-axis is the exon rank and the x-axis has boxplots of GERP scores for each exon. Exons with mean GERP scores above 0 are colored red and those below zero are colored blue. One can clearly see the GERP score reduce considerably between exons 160 and 200.

Runs of short exons in Titin gene are conserved across all Mammals

Titin gene more commonly known as Connectin is an elastic muscle protein. The complete gene sequence for this gene has been known since 2001. 

Here we compare the exon structure of Titin transcripts across multiple species with special focus on the cluster of short exons(< 100 bp) in the middle of the gene. Exon ranks of these exons is from ~100 to 180.  These exons correspond to the PEVK segments that are thought to provide the protein "spring" like properties. Comparison of the Titin gene across multiple species has previously been performed across large evolutionary distances. 

All four rodent species [Kangaroo rat (Dipodomys ordii), Mouse (Mus musculus), Pika (Ochotona princeps) and Rabbit(Oryctolagus cuniculus)] from Ensembl show the PEVK exons in phase 1 followed by a pair of 3 exons as seen the human titin gene. 

Mouse

Rabbit

Kangroo Rat

Pika

Similarly, all 12 species from Laurasiatheria[Alpaca (Vicugna pacos), Cat (Felis catus),Cow (Bos taurus), Dog (Canis lupus familiaris), Dolphin (Tursiops truncatus), Ferret (Mustela putorius furo), Hedgehog (Erinaceus europaeus), Horse (Equus caballus), Megabat (Pteropus vampyrus), Microbat (Myotis lucifugus), Panda (Ailuropoda melanoleuca), Sheep (Ovis aries)] also have the PEVK cluster.

Alpaca

Cat

Cow

Dog

Dolphin

Ferret

Horse

Megabat

Microbat

Panda

Sheep

The European Hedgehog has only 30 exons annotated in Ensembl release 78. This might change in later releases. So Functional characterization or some other form of validation is required before considering the possibility of the loss of large number of exons.

All 8 primate genomes [Chimpanzee(Pan troglodytes), Gibbon (Nomascus leucogenys), Gorilla (Gorilla gorilla gorilla), Marmoset (Callithrix jacchus), Olive baboon (Papio anubis), Orangutan (Pongo abelii), Tarsier (Tarsius syrichta) and  Vervet-AGM (Chlorocebus sabaeus)] are very similar to the Human titin. The Gibbon TTN gene is not showing the characteristic run of short exons, but this could be due to the Gibbon genome being relatively new.

Gorilla

Chimp

Gibbon

Marmoset

Olive babbon

Tasrsier

Vervet-AGM

Orangutan

While we have looked at birds before, it has not been a complete coverage. Moreover the ortholog for the TTN gene has been named the SPEG complex locus. So here we use the SPEG complex locus transcripts instead of the transcript from the gene annotated as Titin. We can see that the Chinese Turtle (Pelodiscus sinensis) shows the characteristic pattern of the PEVK exons. However, none of the transcripts annotated in birds [falbicollis_ENSFALG00000003453,ggallus_ENSGALG00000028386,acarolinensis_ENSACAG00000013938,mgallopavo_ENSMGAG00000011306 tguttata_ENSTGUG00000006089]

Chinese Turtle

show the pattern.


Another post dealing with Fish, insects and other species might be able to find other interesting changes in the Titin gene during the course of evolution.

Titin gene in Fugu - 3 copies or annotation and assembly error?

My growing obsession with the TITIN gene is hopefully not a sign of something else.

The Fugu genome was sequenced, assembled and released soon after the human genome. As was customary during the release of genomes at the beginning of the genomic era it was released with much fanfare. It was flaunted as a good model for comparing synteny with the Human genome. Analysis of the Fugu genome has also been used to find evidence for whole genome duplication in Ray-Finned fish. 

Titin has two copies, ENSTRUT00000001472 flanked by enox1 and AIM29 [Altered Inheritance rate of Mitochondria] (annotated as 1 of 2) and ENSTRUG00000003764 flanked by TTNB and ubiquitin-conjugating enzyme E2G 2 (TTN 2 of 2). So this suggests the Fugu genome has 3 copies of the Titin gene, if one considers the TTNB gene as the third copy. 

Existence of two adjacent copies of the Titin gene in Zebrafish has been validated by multiple methods. This can also be seen reflected in the latest release of the assembly. TTNA (ENSDARG00000028213) and TTNB (ENSDARG00000000563) are atleast 93 and 82 Kb long. 

The African clawed frog has a different view of things with the TTN gene (ENSXETG00000015021) followed by the TN (Titin Novex-3 or ENSXETG00000024421) gene. While the TTN gene is ~94 Kb long, the Tn gene is annotated as a paralog and has a length of ~15Kb. 

A rather complex history of duplication, retention and loss of this gene which also happens to be the longest known gene makes its annotation and study all the more difficult. Functional characterization of the various orthologs, paralogs and isoforms might be a way forward to understand and distinguish assembly/annotation errors from real biology. 

Largest known protein - Titin

With a length of more than 30Kb Titin is the largest known protein in the Human genome. Due to its repeated use of the same domains, it is thought have undergone very unpredictable exon losses. 

Apart from the biological reality of exon loss, the incredible length of the gene makes it prone to annotation errors. Prevalence of such large scale annotation errors makes it impossible to study the intricacies of the biology of such a gene. This is an attempt to identify such potential errors in annotation. The hope is that it will contribute to improving the annotation. 

Mutations in the Titin gene have been implicated in many diseases. Being the longest gene also makes it interesting from an evolutionary point of view. 

Chicken:

The human version of the gene (located on chr-2) has 363 annotated exons as per Human release 75 of Ensemble. The chicken version of the gene (located on chr-7) has only 47 annotated exons as per Chicken release 75 of Ensemble. Flanking genes are PLEKHA3 and CCDC141. However, a new gene, "ENSGALG00000026366" has been annotated in Chicken between PLEKHA3 and TITIN. While it has a name like "gga-mir-7474" and a link to the mirbase, it has gene type as protein coding. If that was not confusing enough, this gene has an aminoacid length of ~30Kb (has 269 exons). Given its location and length, it appears that the Titin gene has been incorrectly split into two genes (Titin itself and gga-mir-7474). As expected, the two genes are connected by a chicken cDNA EST (see figure below).

This "gga-mir-7474" gene gets top blast hit from Titin. 

So based on EST data and blast data these two genes can be merged as part of the Titin gene. We are still short by 47 more exons.

Flycatcher:

The release 75 of Ensemble has a ~61Kb long TTN gene with 106 exons annotated in Flycatcher. Two very short genes (with 1 and 2 exons) downstream from this TTN gene, ENSFALT00000015943 and ENSFALT00000003626 also give top blast hit to the TTN gene.

Even with availability of EST evidence, some very good objective predictions, the annotation seems rather sketchy.