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)