Presented by: Matthew Tippin, Bianca Sanchez Mora RNA Maps Reveal New RNA Classes and a Possible Function for Pervasive Transcription Philipp Kapranov1, Jill Cheng1, Sujit Dike1, David A. Nix1, Radharani Duttagupta1, Aarron T. Willingham1, Peter F. Stadler2, Jana Hertel2, Jörg Hackermüller3, Ivo L. Hofacker4, Ian Bell1, Evelyn Cheung1, Jorg Drenkow1, Erica Dumais1, Sandeep Patel1, Gregg Helt1, Madhavan Ganesh1, Srinka Ghosh1, Antonio Piccolboni1, Victor Sementchenko1, Hari Tammana1, Thomas R. Gingeras1,* Presented by: Matthew Tippin, Bianca Sanchez Mora

Background Multiple RNA’s of various lengths have already been discovered Idea of pervasive transcription existed Large amount of transcription in genome far beyond only protein genes No known function for ncRNA (miRNA, piRNA, snoRNA, siRNA etc) Believed to be an evolutionary relic of RNA world Purpose of experiment was to investigate lRNA and sRNA

Affymetrix Microarray

Tiling Array 5bp 35bp Mismatch/Perfect Match differ by one bp (unstable) Mismatch: negative control, primer not bound correctly Perfect Match: binds correctly HG30 – genome transcript Probe pairs represent 84% nt of the repeated sequences Included LINE2 35 bp resolution Probe pairs represent 91% of the repeat mass sequence with at least 5 probes

Generation of RNA Maps Generation of single-sample RNA graphs Each sample represented by a set of 24 graphs per chromosome (HC version 35) Separate signals by chromosome integrate to create chromosome wide signal value Generation of sRNA transcript maps HepG2/HeLA cells – hybridize to either +/- strand resolution bp sets Generation of lRNA transcript maps Transcripts > 200 nt mapped from cytoA+ RNA from 8 lines – 2 nuc 3 replicas repaired, composite signal generated connect adjacent positive probes; discard transfrags overlapping pseudogenes/low complex/repetitive seq

Analysis of sRNA Conserved Long Nuclear Transfrags that Overlap sRNAs are enriched near TSS Short RNAs can associate with lRNA transripts not detected by transfrags Human-Mouse syntenic analysis of sRNAs 7 nt resolution of several human and mouse syntenic regions were interogated

qtPCR Analysis of lRNA + sRNA U6: used as a control Power SYBR greenmaster mix (applied BioSystems) Gene specific primers used for qRTPCR using one step method where reverse transcription is performed in the same tube as subsequent qPCR Triplicate reactions were set up for every primer pair Analysis of expression performed by comparing CT values for a series of nonspecific control genes with primers for genes of interest

Subcellular distribution of nuclear and cystolic RNA classes

Conserved sRNA Surrounding exon of RYR3 gene Phastcon: computer algorithm program that shows conservation RYR3: gene that codes for ryanodine receptor (releases Ca2+ from storage) Phastcon: program that shows conservation Scores indicate 1/5 sRNA transfrags were evolutionarily conserved

Comparing Phastcon scores of overlapping and nonoverlapping lRNA to sRNA

Conservation extends beyond lRNA overlap Indicates regions outside of overlap may be significant

sRNA + lRNA Overlap More sRNA transfrags overlap with nuclear lRNA transfrags as opposed to lRNA overlapping sRNA lRNA with overlapping sRNA are more conserved than lRNA without

PALRs sRNA overlap lRNA PALRs: promoter-associated lRNAs lRNA that overlap 5’ end of protein coding genes Map the same region as PASRs

PASRs and TASRs sRNA clusters – 3’ and 5’ ends of genes Northern Blot: Detects RNA to study gene expression PASRs: Promoter-associated sRNA TASRs: Termini-associated RNA PASRs expressed at similar lvl to genes they overlap

Frequency Distribution via Violin Plots Positive Correlation shown between Density PASRs + Expression level sRNA have biological significance

Distribution of syntenically conserved sRNAs PASRs and TASRs are most conserved sRNA Conservation between species Northern Blot show comparable size

Summary of Results Overall the results indicate: lRNA act as primary transcripts for sRNA There are three distinct classes of RNA found Possibly 10% ncRNA appears to have gene regulatory function that was previously unknown

Conclusion lRNA can be primary transcripts for the production of sRNA Three novel potentially functional classes of RNAs were identified (2 are syntenically conserved and correlate with the expression state of protein-encoding genes) Data supports a model of genome organization – RNA regulation in gene expression

Significance Classification of 3 different RNA classes lRNA and sRNA relationship suggests complex network of ncRNA regulation of gene expression in higher eukaryotes Regulation possibly due to chromatin remodeling complexes Expands Central Dogma Future insights into the human transcriptome

Additional Reading Supplemental Reading: Clarify methods, data collection and analysis Kapranov et al, (2007) Supplemental Reading www.sciencemag.org/cgi/content/full/1138341/DC1 Later Study by Kapranov (2010): A continuation of the idea brought forth Most nuclear non rRNA is unannotated Kapranov et al; BMC Biology, 2010, 8:149, doi: 10.1186/1741-7007-8-149.

References ENCODE-Project-Consortium, Nature J. Cheng et al., Science 308, 1149 (2005). P. Kapranov et al., Science 296, 916 (2002). Materials and methods, Science online