the transcriptional landscape of the mammalian genome riken and fantom consortium carninci, et al...

The Transcriptional Landscape of the Mammalian Genome

RIKEN and FANTOM Consortium Carninci, et al (2005)

Chris Chander, Luke AdeaBioSci D145

Feb. 12, 2015

How many “genes” do we have?

● Do humans only have 30,000 genes?● Raises the question of what constitutes a gene● There is a significant portion of the genome that

does not encode protein- what transcripts are derived from those

regions?

Why do we want to analyze RNA transcripts?

● Identify protein-coding transcriptso as well as function of noncoding RNAs (ncRNAs)

● Understand transcriptional regulationo both in differentiation and development

● Understand transcript conservation○ if sequence is conserved than maybe it has

relevance

The Transcriptional Landscape

● Pattern of transcriptional control signals and the transcripts generated

Ditag Technologies

● Ditags are short sequences at the 5’ and 3’ ends of a DNA fragmento Gene identification signature (GIS) o Cap-analysis gene expression (CAGE)o Gene signature cloning (GSC)

allows acquisition of rare genes

Approach

● Combined full length cDNA isolation with ditag technologies● to identify initiation and termination sites

Cap-Analysis Gene Expression (CAGE)

Methods

● 1 million CAGE tags produced from 2 HepG2 CAGE librarieso one constructed with random primerso the other with oligo-DT primers

● CAGE tags mapped to genome○ identified likely promoters ○ transcription start sites (TSS)○ genomic span of primary transcript

Genome-Transcriptome Relation

● Full length cDNAand GSC ditags distribute together● Mega-transcriptsfound at upper end ofdistribution

Transcriptional unit vs transcriptional framework

● Transcriptional unit (TU)o mRNAs that share at least one nucleotideo same genomic locationo same genomic orientationo However, TU fusion can join unrelated transcripts

Transcriptional unit vs transcriptional framework cont...

● Transcriptional framework (TK)○ group of transcripts that share

■ common expressed regions■ splicing events■ transcriptional start sites■ termination events

Genome has much more transcription than expected

● TKs are closely associated in what is called transcriptional forests (TF).

● Transcription in TFs occurs without gaps and can occur on either strand.o vary in RNA size some up to 1MB

● Based on the number of transcripts produced from the CAGE method there is an order of magnitude more transcripts than “genes” in mice

● Genome tiling arrays suggest 10x more transcripts encoded than the number of “genes” in humans

Transcript diversity in start, splicing and termination sites● Transcription initiation can

occur in any region of a gene.

● 65% of TU contain multiple splice variants

● Alternative termination sites discovered via analysis of transcripts 3’ ends

Intergenic distances

● Compares distance between TUs● Shorter distance between genes using tail to tail (3’ end of

one strand to 3’ end on antisense strand) configuration● Suggests antisense regulatory mechanism for downstream

genes

Conservation of Promoters

● Promoter regionsof ncRNAs are moreconserved than coding RNAs ● Suggests that ncRNAs display positional conservation

Implications

● Suggests much more transcription and transcript diversity than previously thought○ numerous transcript variants in one gene○ at least 10 times more transcripts than number of genes

● Will lead to further study of ncRNA function○ ncRNA contain multiple regulatory elements

● Transcription occurs on both strands ○ Genome manipulation in mice may affect more than one

TK

Critiques and Limitations

● Extensive use of acronyms and new terminology

● The figure legends do not explain the graphs clearly- ambiguous color schemes

Further Reading

● Long noncoding RNA function, NEAT 1Imamura, K. Long noncoding RNA NEAT1 dependent SFPQ relocation from promoter region to paraspeckle mediates IL8 expression upon immune stimuli. 2014. Mol Cell. 6;53(3):393-406

Future Directions

● Further characterize functions for ncRNA ● Establish a more encompassing definition of

a gene (that includes or distinguishes ncRNA)