COMPUTATIONAL GENOMIC STUDY OF THE CO-TRANSCRIPTION OF THE GENES UBE2V1 AND Kua

Alicia Giménez Hidalgo (alicia.gimenez01@campus.upf.edu)
Gemma Guevara Manjón (gemma.guevara01@campus.upf.edu)

Facultat de Ciències de la Salut i de la Vida

Universitat Popeu Fabra

ABSTRACT

Genes UBE2V1 and Kua can be co-transcribed, generating the fusion protein Kua-UEV. UBE2V1 gives the hybrid protein its B domain and function, whereas Kua provides the A domain and the cytoplasmic location. Kua-UBE2V1 is a variant of Ubiquitin-conjugating enzyme E2; it seems to be involved in noncanonical ubiquitination, and modulates c-FOS transcription in mammary cells. The three genes give rise to several different transcripts and isoforms, and orthologues can be found in other species. The genes are highly expressed in blood cells and immunitary cells as well as in the respiratory system.

INTRODUCTION MATERIALS & METHODS RESULTS CONCLUSION

INTRODUCTION

Ubiquitinylation, the post-translational attachment of ubiquitin to proteins, alters the function, location or trafficking of a protein, or targets it to proteasome degradation. The process involves the sequential action of at least three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). The E1 enzyme mediates an ATP-dependent transfer of a ubiquitin molecule to a cysteine residue on the E2 enzyme. The E2 enzyme then either transfers the ubiquitin moiety directly to a substrate, or to an E3 ligase, which can also ubiquitinylate a substrate.
There are several different E2 enzymes (over 30 in humans), which are broadly grouped into four classes, all of which have a core catalytic domain (containing the active site cysteine), and some of which have short N- and C-terminal amino acid extensions, which appear to be important for localisation and protein-protein interactions. In addition, there are variant proteins with an E2-like fold that are devoid of catalytic activity, but which appear to assist in poly-ubiquitin chain formation.

The aim of this report is to realize a computational genomic study of human UBE2V1 (Ubiquitin–conjugating Enzyme E2 Variant 1) and Kua, two independent genes contiguously located in the same chromosome that can be co-expressed. So, UBE2V1 and Kua can be expressed as two separate transcripts encoding for two different proteins or be co-expressed in a transcript coding for the two-domain fusion protein Kua-UBE2V1.

It is know that UBE2V1 is a nuclear protein, whereas both Kua and Kua–UEV localize in cytoplasm. This indicates that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination.

In this report we will study the co-expression of these genes, as well as the expression of both genes separatedly.

MATERIALS & METHODS

In order to write the report, we have used different resources.

GENE STRUCTURE: data was obtained mainly from NCBI and UCSC Genome Browser. However, part of the information of this section from UBE2V1 was obtained from ENSEMBL.
HOMOLOGY STUDY: data for UBE2V1 is from ENSEMBL, whereas information about Kua and the hybrid Kua-UEV is from NCBI. A phylogenetic tree has been done using protein sequences extracted from NCBI and program CLUSTALW.
GENE EXPRESSION: this data is from UCSC Genome Browser and VisiGene.
GENE FUNCTION: for this section we used information from Gene Ontology and some published articles (Pubmed search).
PROMOTER REGION: the study of the promoter region has been done in two ways:
- PERL program
- PROMO web server:
  - Get gene promoter region (1kb upstream and 100bp downstream from TSS) from UCSC.
  - Introduce promoter sequence in the PROMO web server.
  - Get data of transcription factors predicted within a dissimilarity margin less or equal than 15%.
  - Filter to keep only binding sites/TFs with RE-equally smaller than 0,1.

RESULTS

Here we show the results of the separate study for the two genes UBE2V1 and Kua and the fusion gene Kua-UEV. They include the characterization of the genomic structures, the homology of the genes in other species, the gene expression and its function. Besides, a study about the promoter region and transcription factors that might bind to it has been done.

Fig.1 On the top part there is a representation of the transcripts of the two genes as well as the fusion of the genes. In the mid-table there is the conservation of the hybrid gene.

The results will be studied separatedly. Click the following links in order to see the results for each gene:

KUA-UBE2V1 UBE2V1 KUA

CONCLUSION

The hybrid gene Kua-UEV results from the fusion of two adjacent genes, Kua and UBE2V1, located in chromosome 20q13.2, in strand minus. These genes can either codify for two independent proteins or for the fusion protein Kua-UEV which mantains the function of UEV but in a different cellular location.

UBE2V1 is a variant of the subfamily of ubiquitin-conjugating E2 enzymes, wich encodes for inactive E2 proteins. This gene has five transcript variants generated by alternative splicing that codify for four isoforms. The resulting proteins have between 103 and 170 aa, and are located in the nucleus. Besides, the upstream gene Kua is a newly discovered pseudogene which only has one transcript that encodes for a 267aa long protein and for whom there is no information about its function. The fusion of both genes generates the variant Kua-UEV where domain B comes from UBE2V1 and the A domain comes from Kua. This gene has two transcripts and two isoforms, one of 221aa and the other of 370aa. These proteins are known to mantain the UBE2V1 function but a cytosolic localization. Because the sequence of aminoacids is the same in all the variants, we asume that there is no reading-frameshift in any of the genes' transcripts.

The two genes Kua and UBE2V1 have been well conserved throughout evolution because of a positive selection pressure. There is a high conservation in those species that are more phylogenetically similar, and less in those that are more different.

According to the expression, the three genes (Kua, UEV and the fusion Kua-UEV) are observed to be highly expressed in blood cells and immunitary cells as well as in the salivary glands and the respiratory system (like lungs or bronchial epithelium). There is evidence that the three are underexpressed in brain structures like the amygdala or the caudate nucleus. However, there are organs such as pancreas or ovaries where the expression can differ depending on the gene.

Because the gene Kua has no known function, and the hybrid Kua-UEV has the same function as UEV but in a different localization we should only mention the function of UEV. The protein encoded by this gene is located in the nucleus and regulates noncanonical elongation of ubiquitin chains, a variant of polyubiquitination. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. The overexpression of UEV1 in human colon cancer cells induces the accumulation of cells in G2-M and poliploidy, apoptosis, and inhibition of cell differentiation.

Regarding characterization of the genes' promoter regions, first of all we can conclude that of the two methods used, the best one is PROMO server, because the bioinformatics are much more accurate. Fusion Kua-UEV and Kua have the same promoter region, and thus the same TFs. The coincident TFs between the two methods have been: NF-AT1, NF-kappaB and RXR-alpha for Kua-UEV and Kua and NF-AT1, NF-kappaB, YY1, RXR-alpha, AhR and HNF-4 for UBE2V1.

Finally, we can conclude that biology is not as simple as it seems at first sight -one gene, one protein-, since gene products can also be expressed as fused proteins, giving more complex functions to cells. In our subject of study (Kua-UEV fusion) in order to obtain more information about the genes, more research on Kua should be carried.

REFERENCES

- Thomson TM, Lozano JJ, Loukili N, Carrio R, Serras F, Cormand B, Valeri M, Diaz VM, Abril J, Burset M, Merino J, Macaya A, Corominas M, Guigo R. Fusion of the human gene for the polyubiquitination coeffector UEV1 with Kua, a newly identified gene. Genome Res 2000 10: 1743-1756
- Hofmann RM, Pickart CM. MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair.Cell. 1999 Mar 5;96(5):645-53.
- Sancho E, Vila MR, Sanchez-Pulido LRole of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells.Mol Cell Biol. 1998 Jan;18(1):576-89

Databases and Software

- NCBI: http://www.ncbi.nih.gov/
- Ensembl: http://www.ensembl.org/
- Clustalw: http://www.ebi.ac.uk/clustalw/
- UCSC: http://genome.ucsc.edu/
- PROMO 3.0: PROMO homepage