PEIJ_PIEJ DB: A Putative Database for Intron Retention

doi:10.21203/rs.3.rs-1979709/v1

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PEIJ_PIEJ DB: A Putative Database for Intron Retention

https://doi.org/10.21203/rs.3.rs-1979709/v1

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Unlike exon skipping, intron retention is another type of important mechanism of alternative splicing in higher eukaryotes. Although high throughput tandem (MS/MS) mass spectrometry provides extensive information for rapidly identifying potentially novel alternatively-spliced protein products from experimental datasets, the possibility to identify alternative splicing events through tandem mass spectrometry relies on the database with which the spectra will be searched. In this research, the previous putative exon-exon junction database (PEEJ DB) aimed at and specified for putative exon skipping events in all kinds of alternative splicing studies is extended and a theoretical exon-intron junction and putative intron-exon junction database (PEIJ_PIEJ DB) to account for all possible exon-intron (or intron-exon) combinations and all possible intron internal retention events for every gene in the human genome from the EnsEMBL core database has been built up.Application of the constructed PEIJ_PIEJ DB to non-ETS gene fusion sequences in human prostate cancer RNA-Seq data demonstrates the putative exon-intron junction and putative intron-exon junction database (PEIJ_PIEJ DB) can identify intron retentions with reliable confidence in the non-ETS gene fusion sequences, suggesting that this putative database is helpful for scientific researchers in alternative splicing studies involving intron retention events from high-throughput experimental datasets such as proteomics datasets and next generation sequencing ones. The sourcecode implementing the database is freely available at https://sourceforge.net/projects/peij-piej.

Alternative splicing in eukaryotes is an important gene regulation mechanism. It has been estimated that ~ 95% of intron-containing genes in humans and over 60% of multi-exon genes in plants are alternatively spliced [1, 2]. Previous studies have shown that, with the advantage of mass spectrometry (MS) and rapidly accumulating high-throughput MS/MS (tandem MS)-based proteomics data, MS-based peptide sequence data can be mined to identify and validate alternative splicing events of genes in a genome-wide manner. However, the ability to identify alternative splicing events through tandem mass spectrometry depends on the database against which the spectra would be searched [2].

Besides of exon skipping [2, 3], intron retention is also a typical type of important regulating mechanism of alternative splicing [4].

In this research, the previous putative exon-exon junction database (PEEJ DB) [2] which was specified for exon skipping events has been extended and a putative exon-intron and putative intron-exon junction database (PEIJ_PIEJ DB) from EnsEMBL Core Database specified for intron-retention events has been described. Application of PEIJ_PIEJ DB to non-ETS gene fusion sequences in human prostate cancer RNA-Seq dataset [8] demonstrates the putative exon-intron junction and putative intron-exon junction database (PEIJ_PIEJ DB) has identified and revealed several intron retentions with high confidence in the non-ETS gene fusion sequences, manifesting that this database could assist scientific researchers in alternative splicing studies involving intron retention events from rapidly accumulating proteomics datasets (for example, mass spectrometry ones) and next generation sequencing datasets (for example, RNA-Seq ones).

2.1. Construction of the Putative Exon-intron Junction and Putative intron-exon Junction Database from Ensembl Core Database

Typically, there are three classes of mass spectra (or RNA-Seq reads) that represent possible intron retention events(Figure 1): (A) A mass spectra (or an RNA-Seq read) that maps to an exon-intron junction; (B) A mass spectra (or an RNA-Seq read) that maps to an intron-exon junction; (C) A mass spectra (or an RNA-Seq read) that maps to an intron only (intron internal).

The EnsEMBL Core Database (homo_sapiens_core_89_38h) is downloaded from the Ensembl website http://www.ensembl.org into a local MySQL database. The putative exon-intron and putative intron-exon junction database (PEIJ_PIEJ DB) is constructed for the three kinds of mass spectra (or RNA-Seq reads) that represent possible intron retention events.

The putative exon-intron and putative intron-exon junction database (PEIJ_PIEJ DB) is a protein database, which is constructed in details as below:

(1) For Class A intron retention events, entries are presented as 50AA sequences (25 amino acids are taken which are translated from the end of an exon and connected to 25 amino acids which are translated from the beginning of the following intron).

(2) For Class B intron retention events, entries are presented as 50AA sequences (25 amino acids are taken which are translated from the end of an intron and connected to 25 amino acids which are translated from the beginning of the following exon).

(3) For Class C intron retention events, since the strand information is known in advance, a three-frame translation approach which is similar to six-frame translation [45] is adopted to exhaustively account for all possible in-frame [4] and frame-shift [21] and partial intron internal retention events [29, 46-48].

The reason for choosing 50 amino acids for an entry in PEIJ_PIEJ DB for Class A and Class B intron retention events is described as in the PEEJ DB [2]. Briefly, a typical ion-trap mass spectrometer has a window size to detect peptides with molecular weight from 500 to 3000 daltons. A peptide of 25 amino acids has a molecular weight of about 3000 daltons, covering the upper range of MS detection [2].

As with the PEEJ DB [2] for exon skipping events, the phase information and strand information of all the exons in the human genome in the ensembl database has also been considered in the construction of the putative exon-intron and putative intron-exon junction database (PEIJ_PIEJ DB) in this research.

Table 1 depicts the database structure of the putative exon-intron and putative intron-exon junction database (PEIJ_PIEJ DB).

Table 1. Database Structure of PEIJ_PIEJ DB

Database Schema

Description

Entries for Class A intron retention

events

25 amino acids are taken which are translated from the end of an exon, 25 amino acids are taken which are translated from the beginning of the following intron, then connecting the two parts

Entries for Class B intron retention

events

25 amino acids are taken which are

translated from the end of an

intron,25 amino acids are taken

which are translated from the

beginning of the following exon,

then connecting the two parts

Entries for Class C intron retention

events

three-frame translation of the

intron sequences to account for all

possible in-frame, frameshift and

partial intron internal retention

events

The database is implemented in perl, Bioperl, mysql and Ensembl API which a fasta file with 4,280,722 entries, with gene symbol, chromosome, strand, exon symbol, exon phase information and junction position clearly defined and the Perl source code which implemented the database is freely available at https://sourceforge.net/projects/peij-piej. It should be noted that although the database is based on annotations from ENSEMBL (homo_sapiens_core_89_38h), in the related studies, users can easily modify the script which implements the database to update the database when new ENSEMBL build and new annotations are available to include new information (new intron retention events).

2.2. Utility

Like the putative exon-exon junction database [2, 3] which is specified for exon skipping events, researchers can search against the PEIJ_PIEJ DB for known and novel intron retention events from mass spectrometry data using the X!Tandem open source protein identification program [5] or the TurboSEQUEST [6] (for a workflow with mass spectrometry data, users can refer to Mo et al (Mo et al., 2008) by simply replacing the PEEJ DB with the PEIJ_PIEJ DB constructed in this study). In addition, when RNA-Seq data is available, researchers can also align the RNA-Seq reads to the PEIJ_PIEJ DB for intron retention events (known or novel) identification (for a workflow with RNA-Seq data, users can refer to [7] by simply replacing the PEEJ DB with the newly constructed PEIJ_PIEJ DB).

3.1. Case Study

To demonstrate the utility of the putative exon-intron and putative intron-exon junction database (PEIJ_PIEJ DB), in this study, the mRNA sequences of the novel non-ETS gene fusions in human prostate cancer in an next-generation RNA sequencing dataset [8] are utilized.

The mRNA sequences of the novel non-ETS gene fusions in human prostate cancer cells in the next-generation RNA sequencing dataset [8] are downloaded from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) with accession numbers HM245385–HM245396. The sequences are then searched against the putative exon-intron and putative intron-exon junction database (PEIJ_PIEJ DB) using the BLASTX program [9, 10] with default parameters.

Figure 2 shows a high confidence (98% identities for the BLASTX program) intron retention occurred in the human ALG5-PIGU breakpoint junction mRNA sequence. Searching with the UCSC Human Genome Browser [11] shows that ALG5 is a dolichyl-phosphate beta-glucosyltransferase, and PIGU is participated in phosphatidylinositol glycan anchor biosynthesis in the cells.

Figure 3 shows a high confidence (86% identities for the BLASTX program) intron retention event which is occurred in the human KLK2-ETV1 breakpoint junction mRNA sequence. KLK2 is a member of the human kallikrein gene family [12–16]. Searching with the UCSC Human Genome Browser [11] shows that ETV1, which is also known as ETS variant 1, has 23 transcripts. Previous studies show that intron retention is a common splicing phenomenon in the human kallikrein gene family [17]. The first six centromeric members of the kallikrein (KLK) gene family have splice variants that retain intron III [17].

The 12 mRNA sequences of the known and novel non-ETS gene fusions in human prostate cancer [8] and the BLAST analysis result for all left anchoring and right anchoring of IR (intron retention) for the 12 mRNA sequences in human prostate cancer in the next-generation RNA sequencing dataset are presented in Supplementary Files 1–2 respectively. Besides of the two examples mentioned above, it can be found that several other high confidence intron retention events (left anchoring and right anchoring of IR) in the novel non-ETS gene fusion junctions are also revealed by the PEIJ_PIEJ DB in this study.

3.2. Compare with SASD

PEIJ_PIEJ DB outperforms SASD [18] in terms of intron retention identification. For example, searching by ENSG00000091157 in SASD yields 1,104 alternative splicing peptides in which 155 are exon-intron or intron-exon junctions. And PEIJ_PIEJ DB contains 430 possible intron retention events for the gene ENSG00000091157. The intron retention events captured by PEIJ_PIEJ DB but not by SASD mainly correspond to those that are frame-shift intron retentions [21] and partial intron retentions [29, 46–48] which in theory are putative and some of which are experimentally verified by others.

Another advantage of PEIJ_PIEJ DB over SASD [18] is that users can search against PEIJ_PIEJ DB directly with their mass spectrometry or RNA-Seq datasets. However, SASD currently does not have such a function.

Intron retention is an important subject to understand several biological processes in higher eukaryotes. It is a common cause for cancer [19–42]. It's also a common phenomenon in Arabidopsis [43, 44]. Zhang et al probed the global landscape of intron retentions in lung adenocarcinoma using RNA-Seq technology and an abundant of intron retention events were found in both tumor and normal tissues. Combined with gene expression analysis, they found that genes with tumor-specific retentions tend to be over-expressed in tumors, and the abundance of intron retention within genes is negatively related with gene expression which suggests the action of nonsense mediated decay [4]. Middleton et al found that IR corresponds to lower protein levels and intron-retaining transcripts which escape nonsense-mediated decay are not translated actively [49].

In this study, a theoretic putative exon-intron and putative intron-exon database (PEIJ_PIEJ DB) to account for all possible exon-intron (or intron-exon) combinations and all possible intron internal retention events for a gene from the EnsEMBL core database is built up. Besides of in-frame intron retention events, PEIJ_PIEJ DB can also capture frame-shift [21] and partial intron retention events [29, 46–48].

As is shown in the case study in this research, some gene fusion events are tightly coupled with intron retention events with high confidence in the BLAST analysis [9, 10] in human prostate cancer, suggesting some gene fusion phenomenons are ultimatedly mediated by some intron rentention mechanisms in human prostate cancer, which also suggests that the constructed PEIJ-PIEJ DB can assist scientific researchers in alternative splicing studies involving intron retention events from high-throughput experimental datasets in an intelligent manner in biomedical researches.

The caveat of PEIJ_PIEJ DB is that it is based on annotations from ENSEMBL core database (for an example, homo_sapiens_core_89_38h). It is constrained to the identification of intron retention events which are derived from combinations of known exon-intron as well as intron-exon junctions and the products from three-frame translation of the introns. It is not allowed for the identifying of intron retention events which would have involved in un-annotated exons (or introns) in the human genome. When new ENSEMBL build is available, users need to modify the script which implemented the database to update the database to include new information (new intron retention events) accordingly.

Funding

This article is funded by a research founding granted by the Systems Biology Division, Zhejiang-California International Nanosystems Institute of Zhejiang University, Hangzhou, China.

Availability of data and materials

Project home page: https://sourceforge.net/projects/peij-piej

Operating systems: Linux

Programming language: Perl, Bioperl, MySQL, Ensembl API

License: GPL-3.0 License

Supplementary File 1

The 12 mRNA sequences of the known and novel non-ETS gene fusions in human prostate cancer.

Supplementary File 2

The BLAST result of the 12 mRNA sequences of the known and novel non-ETS gene fusions in human prostate cancer against the PEIJ_PIEJ DB.

Consent to Publish

Not applicable.

Authors' Contributions

XH has conceived the study, implemented the database and wrote the manuscript.

Conflicts of Interests

None Declared.

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No competing interests reported.

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PEIJ_PIEJ DB: A Putative Database for Intron Retention

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Abstract

Figures

1. Introduction

2. Materials And Methods

3. Results

3.1. Case Study

3.2. Compare with SASD

4. Discussions

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

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