AGBT 2010 - Nicole Cloonan - The University of Queensland

Translation-State RNAseq of Human Embryonic Stem Cells using Paired-End Sequencing.

Intro to Stem Cells
* hot topic - potential for cell generating therapies
* Self renewable
* pluripotent
* directable
* tractable

Looking at Extracellular space network.
* molecules that control cell-cell interactions (among others)

The "Plurinet"
* defines the pluripotent status of the cell
* protein-protein interactions
(Muller et al, Nature 455:401-505)

Transcriptional complexity
* 6 transcripts per gene on average
* so how does this affect the plurinet

SOLiD RNA-Seq
* have a pipelien... [too fast]
* done SET and PET.
* 80% of tags map, 194M 50mers, 114M 25mers

Tags that don't map:
* LincRNA, intergene, etc...

PET.
* alternate splicing
* works well if you know what the annotations are.
* with PET, you can build transcript models if you don't have them already - learn more about alt. splice
* can be used for novel exon discovery

Chip-Seq from Ku et al
* Extended Exons. 3' exon extensions can be very long.

[Why is this Chip-Seq?]

Do Virtual Northerns
* Size fractionations
* What you find is that most annotated genes have the right refseq predicted lengths.
* however, some are shorter, some are longer
* Frequency at which tags from a particular library match predicted (based on refseq) vs from RNA data... You do see that some have very different results.

RNA are translated...
* if no signal peptide, cytoplasmic (on free ribosomes)
* if has signal, then it's translated by ribosomes bound to membranes
* use sucrose gradient to separate the two populations
* do PET, (35/75bp reads)
* compare signals in both fractions - they come up well in the predicted fraction.

Novel transcription
* membrane associated RNA have very different proportions of extension (mainly long 3' UTRs) than those in the cytoplasmic fraction

MiRNA biogenesis and mRNA interactions.
* use fractionation to test
* RISC associated with polysomes (which works with fractionation)
* complexes stay together through fractionation
* Long UTRS are enriched for mRNA binding sites

Back to Plurinet
* Complexity is incredibly increased with the extra products and miRNA

Summary:
* PET allows you to reconstruct loci level complexity from RNAseq data
* Size fractionation is useful
* translation state RNAseq allow s the capture of mRNA and miRNA data from polyribosomes
* Transcriptional complexity impacts greatly on interactions.