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Chris Chander, Luke Adea BioSci D145 Feb. 12, 2015

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1 Chris Chander, Luke Adea BioSci D145 Feb. 12, 2015
The Transcriptional Landscape of the Mammalian Genome RIKEN and FANTOM Consortium Carninci, et al (2005) Chris Chander, Luke Adea BioSci D145 Feb. 12, 2015

2 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?

3 Why do we want to analyze RNA transcripts?
Identify protein-coding transcripts as well as function of noncoding RNAs (ncRNAs) Understand transcriptional regulation both in differentiation and development Understand transcript conservation if sequence is conserved than maybe it has relevance

4 The Transcriptional Landscape
Pattern of transcriptional control signals and the transcripts generated

5 Ditag Technologies Ditags are short sequences at the 5’ and 3’ ends of a DNA fragment Gene identification signature (GIS) Cap-analysis gene expression (CAGE) Gene signature cloning (GSC) allows acquisition of rare genes

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

7 Cap-Analysis Gene Expression (CAGE)

8 Methods 1 million CAGE tags produced from 2 HepG2 CAGE libraries
one constructed with random primers the other with oligo-DT primers CAGE tags mapped to genome identified likely promoters transcription start sites (TSS) genomic span of primary transcript

9 Genome-Transcriptome Relation
Full length cDNA and GSC ditags distribute together Mega-transcripts found at upper end of distribution

10 Transcriptional unit vs transcriptional framework
Transcriptional unit (TU) mRNAs that share at least one nucleotide same genomic location same genomic orientation However, TU fusion can join unrelated transcripts

11 Transcriptional unit vs transcriptional framework cont...
Transcriptional framework (TK) group of transcripts that share common expressed regions splicing events transcriptional start sites termination events

12 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. 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

13 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

14 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

15 Conservation of Promoters
Promoter regions of ncRNAs are more conserved than coding RNAs Suggests that ncRNAs display positional conservation

16 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

17 Critiques and Limitations
Extensive use of acronyms and new terminology The figure legends do not explain the graphs clearly ambiguous color schemes

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

19 Future Directions Further characterize functions for ncRNA
Establish a more encompassing definition of a gene (that includes or distinguishes ncRNA)

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