Definition - Why do we need AS analysis? - How do we perform alternative splicing analysis? -
How is AS computation performed? - References
Definition
Alternative Splicing is a splicing process of a pre-mRNA transcribed from one gene that lead to different mature mRNA molecules and therefore to different proteins.
The first example of A.S. was described in viruses and then in cells in 1980 with a gene called IgM (Early et al. - 1980). But this was considered as an anomaly of the normal process which was the dogma “one gene, one protein”, that meant all the exons of a gene are use and useful.
Alternative Splicing has emerged as a major mechanism after the high throughput genome sequencing set up in the 90’s and more especially after the design of tools to perform pairwise alignments of genomic and transcripts sequences.
Recent improvements and better accuracy of alignment tools have demonstrated that the assumed one-to-one mapping from a gene to a protein no longer holds. Instead A.S. seems to be a common process to generate proteins.
- To reconstitute events of creation of peptides/proteins from a gene.
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To discover new exons thus new proteins.
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To understand which are the mechanisms involved in A.S. and how they are triggered and regulated, i.e., to understand how cell, tissue, etc. characteristics may affect gene translation leading to more than one protein [e.g., compare by computation the expression of transcription Factor in tissues (Taneri et al. - 2004)].
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To study and discover proteins involved in the A.S. process (splicesosome). To find specific sites involved in the mechanism of A.S. In particular searching regulation A.S. sites such as ESE, ESS, ISE or ISS, activation/repression mechanisms, and some external factors that can influence the A.S. mechanism by itself.
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To find possible “defaults” or errors in the A.S. events that may be responsible directly or indirectly for diseases.
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To analyze the phylogeny evolution of the A.S. mechanisms.
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To help molecular biologists to find specific primers for one specific transcript.
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To help researchers who need to study experimentally the distribution of specific transcripts in different tissues. This research may be linked with the former one.
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To help researchers study experimentally the AS using microarray to build better probes for their chips.
The above list is a non exhaustive list but over spans several of the main possible purposes of A.S. study.
How do we perform alternative splicing analysis?
Experimental approaches were first the only way to analyze the process. It is a long and expensive process. Today, some computational methods have been designed exploiting sequence information. Identifying relevant and valuable resources, gathering information about transcripts and genomes of differentorganisms, integrating them into a meaningful dataset that can be exploited by scientists requires a lot of time for isolated researchers. To support the community and provide scientists the ability to retrieve all transcripts reorganized from a gene, several databases and services are now dedicated to the A.S. events.
How is AS computation performed?
The analysis is performed following two main steps: An Alignment step and a Clustering step.
The process of alignment is simple and consists of an alignment of a genomic sequence against a transcript sequence. This step is executed with all known transcripts extracted from different databases for an organism. These databases are a collection of transcript sequences such as cDNA, mRNA, EST which are often associated with annotations about the sequences. The more accurate are the annotations the best characterization of transcripts is obtained, resulting in an improved accuracy of A.S. results.
The clustering step immediately follows the alignment step. A cluster normally represents or may be representative of the all intermediate transcripts [from the Pre-messenger-RNA(s) to the mature messenger-RNA(s)] required to obtain one or several functional translated proteins from the same gene. Typically, the more transcripts are obtained, the best cluster accuracy.
Some limitations may be noted. The accuracy of the computation to study A.S. depends mostly on several parameters (Blencowe - 2006), including:
- Relevant data, such as Gene Annotation, extracted from databases and their accuracy. Most of these entries have been submitted by users into databases such as Genbank, and some mistakes may have been made in the entry process.
- Most of A.S. events seem to occur on Pre-mRNA as they emerge from the POLII polymerase complex (Bentley - 2005).
- Because of the RNA instability and techniques used to study them, some information may be lost on certain transcripts such as the 5’ region of a transcript or the 3’ region if the reverse transcription has been incomplete. So transcripts may only represent a part of the reality.
Bentley DL. 2005. Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors. Curr Opin Cell Biol 17(3):251-6.
Blencowe BJ. 2006. Alternative splicing: new insights from global analyses. Cell 126(1):37-47.
Early P, Rogers J, Davis M, Calame K, Bond M, Wall R, Hood L. 1980. Two mRNAs can be produced from a single immunoglobulin mu gene by alternative RNA processing pathways. Cell 20(2):313-9. .
Guttmacher AE, Collins FS. 2002. Genomic medicine--a primer. N Engl J Med 347(19):1512-20.
SCRIPPS-GenomeCenter. 2006. Database of Splicing Variants.
Taneri B, Snyder B, Novoradovsky A, Gaasterland T. 2004. Alternative splicing of mouse transcription factors affects their DNA-binding domain architecture and is tissue specific. Genome Biol 5(10):R75. .
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