Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2014; 18: 526-536 Bioinformatics analysis of Exonic Splicing Enhancers (ESEs) for predicting potential regulatory elements of hTERT mRNA Splicing F. WANG 1, G.-M. CHANG 2, X. GENG 3,4,5 1Department of Neurology, General Hospital, Tianjin Medical University, China 2Department of Clinical Laboratory, General Hospital, Tianjin Medical University, China 3Department of Biochemistry and Molecular Biology, Tianjin Medical University, China 4Tianjin Key Laboratory of Cellular and Molecular Immunology, China 5Key Laboratory of Educational Ministry of China Abstract. – OBJECTIVES : Alternative splicing Introduction of human telomerase reverse transcriptase (hTERT) has an important effect on regulating Telomeres are specialized structures located at telomerase activity. Exonic splicing enhancers (ESEs) are a family of conserved splicing factors the ends of eukaryotic chromosomes, that pro - that participate in multiple steps of the splicing tect linear chromosome ends from unwanted re - pathway. Our aim is to analyze the ESEs for pre - pair or recombination . Progressive telomere dicting the potential regulatory elements of shortening occurs in somatic cells due to incom - hTERT mRNA splicing. plete replication of chromosome ends. Telom - MATERIALS AND METHODS: Enter the FAS - erase, a ribonucleoprotein complex, is capable of TA format of hTERT total sequences or individ - adding telomere repeats to the 3 ’ end, which is ual exon as the input data in the main interface essential for the telomere length maintenance in of ESEfinder3.0 and ESEfinder2.0 program. Ana - 1 lyze the data of output results and compare the germ cells and stem cells . Human telomerase differences between ESEfinder3.0 and ESEfind - holoenzyme consists of three components: a er2.0 program. RNA component (human telomerase RNA com - RESULTS: Five ESEs were predicted in exon 5 ponent, hTR), which serves as a template for to exon 9 of hTERT. They were at position 108 lo - DNA replication., a catalytic subunit of telom - cated in hTERT exon 5, at position 92 located in erase with reverse transcriptase activity (human exon 6, at position 22 located in exon 7, at posi - telomerase reverse transcriptase, hTERT) and tion 73 located in exon 8 and at position 5 located 2,3 in exon 9. There were no differences between ES - the telomerase-associated proteins . Most of the Efinder 3.0 and ESEfinder 2.0 in our case. human tissues that lack detectable telomerase CONCLUSIONS: The identification of these po - activity also lack the expression of hTERT but tential ESEs of hTERT might be helpful for the not hTR and telomerase-associated proteins. design of antisense oligonucleotides, which This suggests hTERT acts as a rate-limiting fac - could modulate hTERT alternative splicing and tor for telomerase activity 4,5 . inhibit telomerase activity. Telomerase activity is under precise control at the transcriptional, translational, and post-trans - Key Words: lational level. hTERT gene pre-mRNA alterna - Exonic splicing enhancer, Telomerase, hTERT, Alter - tive splicing is thought be one of the most impor - native splicing, Transcription regulation. tant mechanisms for regulation of telomerase ac - tivity. Alternative splicing is a process by which the exons of the pre-mRNA produced by tran - Abbreviations scription of a eukaryotic gene are reconnected in multiple ways to form alternatively spliced vari - hTERT = human telomerase reverse transcriptase; hTR = human telomerase RNA component; ASVs = alternative - ants (ASVs) during RNA splicing. ASVs may be ly spliced variants; ESEs = Exonic splicing enhancers; translated into different protein isoforms. Alter - ORF = open reading frame; RRM = RNA-recognition native splicing is a major method of gene regula - motif. tion for many proteins 6,7 . 526 Corresponding Author: Xin Geng, Ph.D, MD; e-mail: [email protected] Bioinformatics analysis of ESEs for predicting potential regulatory elements of hTERT mRNA Splicing The hTERT gene consists of 16 exons and 15 bind to the members of the serine/arginine-rich introns and full length of hTERT mRNA is about (SR) protein family, which function both as general 4.0 kb long 8. hTERT protein contains a telom - splicing factors and as regulators of alternative erase-specific motif and seven reverse transcrip - splicing 14-17 . SR proteins have a motif consisting of tase motifs. To date, seven alternatively spliced one or two copies of an RNA-recognition motif sites in the hTERT mRNA have been identified, (RRM) followed by a C-terminal domain highly en - 18,19 including three deletion sites (α, β, γ) and four riched in argine/serine dipeptides (RS domain) . insertion sites. Therefore, the alternate splicing ESE-bound SR proteins recruit the splicing machin - sites produce a large number of possible combi - ery through their RS domains, and/or antagonize nations , resulting in different hTERT ASVs . Cur - the splicing silencer elements 20,21 . rently, a few hTERT ASVs have been identified hTERT, the rate-limiting subunit, plays a cru - in mammalian cells. However, only the full- cial role in regulating telomerase activity. There - length hTERT mRNA without deletion or inser - fore, the alternative splicing of the hTERT gene tion sites has telomerase activity in vivo . may make significant influence on telomerase ac - Most studies on hTERT ASVs involve dele - tivity at the transcriptional level . In this article, tions at the two main splicing sites. Deletion of we will analyze the ESEs of hTERT to predict the α splicing site leads to a deletion of 36 nu - the potential regulatory elements of telomerase cleotides in exon 6, which removes most of the activity, which might become attractive gene reverse transcriptase (RT) motif without disrupt - therapy targets. ing the open reading frame (ORF). Splicing at the β site (deletion of exons 7 and 8) results in a 182 -nucleotide deletion that causes a premature Materials and Methods termination codon, generating a truncated protein lacking the C-terminal and RT motifs B to E. Principle of ESEfinder Splicing at either site , occurring alone or in com - ESEfinder is a web-based program applied in bination, produces a number of ASVs at different rapid analysis of exon sequences to identify puta - levels and proportions . So far , none of the ASVs tive exonic splicing enhancers (ESE ) responsive to identified retains the telomerase catalytic activity the human SR proteins SF2/ASF, SC35, SRp40 and of the full-length product. Nevertheless, some re - SRp55, and to predict whether exonic mutations cent data suggests that splicing patterns of disrupt such elements. This program was released hTERT may play a role in physiological and by Cold Spring Harbor Laboratory (Krainer ’s Lab pathological regulation of telomerase activity, and Zhang ’s Lab) and has been updated to Version and that ASVs are also possibly involved in other 3.0 since 2007 . The homepage for ESEfinder 3.0 is cellular functions 9,10 . http://rulai.cshl.edu/cgi-bin/tools/ Exonic splicing enhancers (ESEs) are the regula - ESE3/esefinder.cgi?process=home and the home - tors of pre-mRNA alternative splicing 4 and partici - page for ESEfinder2.0 is http://rulai.cshl.edu/tools/ pate in the splicing of constitutive exons 11-13 . ESEs ESE2/ (Figure 1). The ESEfinder 2.0 program Figure 1. The main operating interface of ESEfinder 3.0 ( left ) and ESEfinder 2.0 ( right ). 527 F. Wang, G.-m. Chang, X. Geng searches for sequences that act as binding sites for thresholds)” was checked and the “Send” button four members of the SR splicing enhancer proteins. was clicked to generate output data . In ESEfinder Input sequences are screened for consensus binding 2.0 “Send” button was clicked to generate output sequences for the SR proteins SF2/ASF, SRp40, data directly. SRp55 and SC35, using the SELEX (systematic evolution of ligands by exponential enrichment) method. The program scores the input sequences Results according to fit with the loose consensus sequences. The scores above a default threshold value are pre - Prediction of Each Exon Between dicted to act as binding sites with SR protein and Exon 5 and Exon 9 of hTERT by thus function as ESEs. Increased threshold values ESEfinder3.0 (Table II) of ESEfinder 2.0 program for SF2/ASF (from Predicted by ESEfinder 3.0, the highest scored 1.956) and ESEfinder 3.0 for SRp40 (from 2.670), position for each SR protein in each exon be - SRp55 (from 2.676) and SC35 (from 2.383) were tween exon 5 and exon 9 is presented here. Suc - used in order to minimize false-positive results. In cessfully , we predicted the ESEs at position 108 ESEfinder 2.0, only the high-score values (the val - (from 5 ’ end), which got the highest score in ex - ues above the selected threshold) are represented. on 5 of hTERT, the SR protein was SRSF5 There are two options for output information in ES - (SRp40) and the motif was CCACAGG. In exon Efinder 3.0, one is “Report only the best hit in each 6, the ESEs was at position 92, the SR protein sequence (instead of hits above the thresholds)” and was SRSF6 (SRp55) and the motif was the other is “Report all scores in each sequence (in - TGCGTC. In exon 7, the ESEs was at position stead of hits above the thresholds)” 22,24 . 22, the SR protein was SRSF1 (SF2/ASF) and the motif was CAGCCGT. In exon 8, the ESEs Methods and Procedure was at position 73, the SR protein was SRSF6 Find nucleic acid sequence of hTERT (Gen - (SRp55) and the motif was CGCATC. In exon 9, Bank: AH007699.1 ) from NCBI the ESEs was at position 5, the SR protein was (http://www.ncbi.nlm.nih.gov/) and save the in - SRSF6 (SRp55) and the motif was TACGTC formation in FASTA format . Open the homepage (Table II, Figure 2 to 6). of ESEfinder2.0 and ESEfinder3.0.