One gene – one transcript… mostly

Mar Gonzales Porta, Adam Frankish, Johan Rung, Jennifer Harrow, Alvis Brazma European Bioinformatics Institute European Molecular Biology Laboratory Wellcome Trust Sanger Institute

Analysis of RNA-seq data across different tissues and cell lines reveals that the majority of genes have a single dominant transcript

Mar Gonzàlez-Porta Adam Frankish (Sanger) Johan Rung (EBI) Jennifer Harrow (Sanger) Alvis Brazma (EBI)

To appear in Genome Biology on July 1, 2013 http://genomebiology.com/

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polypeptide

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Figure 1

Central dogma of molecular biolgoy

Gene

Transcript

Spliced transcript

The number of genes in human genome

•  Estimates before the Human Genome Project •  ~100,000 genes

•  After •  ~21,000 genes

•  By comparison •  Yeast ~6,000 genes

•  C. Elegance ~17,000

Central gogma revised – one gene many transcritps and proteins via alternative splicing

In human, there are 21,405 protein coding genes and 141,031 different isoforms (92,581 of which are protein coding) annotated, 17,413 genes have >1 isoform

From RNA-seq and other recent experiments

•  Most human genes have more than one splice-form expressed [Pan et al, Nat Genetics 2008], [Wang et al, Nat Genetics 2008] [Mortazavi, Nat Methods 2008]

•  Several isoforms per gene are often expressed to significant levels either in the same cell type or across different [Wang et al, Nat Genetics 2008], [Tang et al, Nat Methods 2009], [Trapnell et al, Nat Biotechy 2010]

•  Isoform expression is regulated [Waks et al, Mol Syst Biol 2011] but splicing can be noisy [Melamud, NAR 2009]

ENCODE – Nature, September 2012

•  ‘Isoform expression by a gene does not follow a minimalistic expression strategy, resulting in a tendency for genes to express many isoforms simultaneously’

•  ‘[…] alternative isoforms within a gene are not expressed at similar levels, and one isoform dominates in a given condition)’

•  Which is it then?

A fundamental question still remains – are different isoforms of the same gene expressed at similar levels?

Expression level

Isoforms 1 2 3 4 5 6

?

Isoforms 1 2 3 4 5 6

We think this is a fundamental question related to the complexity of transcritpome

Data and methods

Illumina Body Map 16 tissues PE (80M) no replicates HiSeq 2000

ENCODE 5 cell lines PE (40M) technical replicates GAII

•  2 different datasets: 46 samples

•  Three different state-of-art tools for transcript quantification (MISO, Cufflinks and mmseq)

•  Direct evidence from splice junctions •  Simulated data – can the methods distinuish between the two

scenarios? •  All approaches produced very consistent results

Most genes express one predominant transcript

Expression level

79% of cases 56% of cases

2-fold dominance 5-fold dominance

Consistent across all studied tissues

What are the RPKM distributions of first, second, … most abundant isoforms for all genes?

Most genes express one predominant transcript

Most genes express one predominant transcript

We detect a total of 31,902 transcripts expressed above 1 FPKM in at least one tissue and 26,641 of these are major transcripts (ratio 1.12)

There is just over one highly expressed transcript per gene! 85% of transcriptome comes from the dominant transcripts Is it the same dominant transcript in all tissues or different tissues tend to have different dominant transcripts?

Major transcripts tend to be recurrent across tissues

Switch events exist ~ 100 strong switches that change the protein sequence

Some of the major transcripts are non-canonical

AES

Do the dominant transcripts of protein coding genes always code proteins?

•  Only in about 80% cases •  However much more often in cytosol than in nucleolus

•  In nucleolus the retained intron is predominantly located towards the 3’ end of the transcript

•  Is this because we extract mRNA before the splicing is completed or can a retained intron be a form of expression regulation?

Conclusions •  Most genes express one predominant transcript over the

rest

•  ~85% of the mRNA pool comes from major transcripts

•  Major transcripts tend to be recurrent across samples, switch events exist but only a small number of these are likely to express different proteins

•  Despite the transcriptome complexity the central dogma of molecular biology may be closer to the truth than recently believed!

Mar Gonzales Porta

Funding

•  EMBL member countries •  European Commission FP7 grants