Do we know it all?

John L. Rinn and Howard Y. Chang Annu. Rev. Biochem. 2012

The RNA world “Central Dogma” DNA RNA Protein

Xist-X-inactive specific transcript Wesley H. Brooks 2009

Reinius et al. BMC Genomics 2010 Xist-X-inactive specific transcript

Micro RNA discovery

The RNA world “Central Dogma” DNA RNA Protein Scaffold catalytic Regulation Small-RNA

Nature Reviews Genetics 12, December 2011Manel Esteller The RNA world- Non-coding RNA

Small non-coding RNA’s microRNA siRNA snoRNA piRNA Nishida N et al. Clin Cancer Res 2012;18:

The RNA world- Non-coding RNA Long non-coding RNA’s John L. Rinn and Howard Y. Chang Annu. Rev. Biochem. 2012

How can we identify them?

M Guttman et al. Nature (2009) LincRNA- Identification of markers such as K4–K36

How can we identify them? lincRNA K4–K36 domains are conserved in their exons and promoters M Guttman et al. Nature (2009) LincRNAs Known lincRNAs Protein coding genes Random intergenic

Do lincRNAs have function?

LincRNAs function John L. Rinn and Howard Y. Chang Annu. Rev. Biochem. 2012

How can we study lincRNAs function?

Experimental system- mouse embryonic stem (ES) cells. Pluripotent cells Can give rise to three primary germ layers Well characterized regulatory networks Systematic loss-of-function experiments on all lincRNAs known to be expressed in ES Generate five lentiviralbased short hairpin RNAs (shRNAs) targeting each of the 226 lincRNAs previously identified in ES cells successfully targeted 147 lincRNAs and reduced their expression by an average of 75% How can we study lincRNAs function?

Experimental system:

For 137 of the 147 lincRNAs (93%), knockdown caused a significant impact on gene expression How can we study lincRNAs function?

Luciferase reporter gene expressed from the endogenous Nanog promoter How can we study lincRNAs function? Nonog- a key transcription factor that is required to establish and uniquely marks the pluripotent state wild-type ES cells and measured mRNA levels of pluripotency marker genes Oct4, Sox2, Nanog, Klf4 and Zfp42.

This establishes that these lincRNAs have a role in maintaining the pluripotent state How can we study lincRNAs function? >90% of lincRNA knockdowns affecting Nanog reporter levels led to loss of ES cell morphology

Do lincRNAs act in repressing differentiation programs? How can we study lincRNAs function?

Many lincRNAs have a role in regulating the ES cell state including maintaining the pluripotent state and repressing specific differentiation lineages.