Post-transcriptional regulation Nearly ½ of human genome is transcribed, only 1% is coding 98% of RNA made is non-coding

ncRNA Structural 1.rRNA 2.tRNA 3.snRNA 4.snoRNA 5.cleavage: Rnases P & MRP, U3, snR30, etc Regulatory 1.Small siRNA miRNA 2.Long Activator Enhancer silencing

Regulatory ncRNA 1.SiRNA direct DNA-methylation via RNA-dependent DNA-methyltransferase 2. In other cases direct RNA degradation

Post-transcriptional regulation RNA degradation is crucial with so much “extra” RNA mRNA lifespan varies 100x Highly regulated! > 30 RNAses in Arabidopsis!

Post-transcriptional regulation mRNA degradation lifespan varies 100x Sometimes due to AU-rich 3' UTR sequences (DST)

mRNA degradation lifespan varies 100x Sometimes due to AU-rich 3' UTR sequences (DST) Endonuclease cuts DST, then exosome digests 3’->5’ & XRN1 digests 5’->3’

mRNA degradation Most are degraded by de-Adenylation pathway Deadenylase removes tail

mRNA degradation Most are degraded by de-Adenylation pathway Deadenylase removes tail Exosome digests 3’ -> 5’

mRNA degradation Most are degraded by de-Adenylation pathway Deadenylase removes tail Exosome digests 3’ -> 5’ Or, decapping enz removes cap & XRN1 digests 5’ ->3’

Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance 1.Nonsense-mediated each splice junction that is displaced by ribosome

Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance 1.Nonsense-mediated each splice junction that is displaced by ribosome 2.If not-displaced, is cut by endonuclease & RNA is degraded

Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance Non-stop decay: Ribosome goes to end & cleans off PABP

Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance Non-stop decay: Ribosome goes to end & cleans off PABP w/o PABP exosome eats mRNA

Post-transcriptional regulation mRNA degradation: mRNA is checked & defective transcripts are degraded = mRNA surveillance No-go decay: cut RNA 3’ of stalled ribosomes

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into bp

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into bp helicase melts dsRNA

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into bp helicase melts dsRNA - RNA binds RISC

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into bp helicase melts dsRNA - RNA binds RISC complex binds target

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into bp helicase melts dsRNA - RNA binds RISC complex binds target target is cut, then digested by XRN1 & exosome

Small RNA regulation siRNA: target RNA viruses (& transgenes) miRNA: arrest translation of targets created by digestion of foldback Pol II RNA with mismatch loop

Small RNA regulation siRNA: target RNA viruses (& transgenes) miRNA: arrest translation of targets created by digestion of foldback Pol II RNA with mismatch loop Mismatch is key difference: generated by different Dicer

Small RNA regulation siRNA: target RNA viruses (& transgenes) miRNA: arrest translation of targets created by digestion of foldback Pol II RNA with mismatch loop Mismatch is key difference: generated by different Dicer Arrest translation in animals, target degradation in plants

small interfering RNA mark specific targets once cut they are removed by endonuclease-mediated decay

Most RNA degradation occurs in P bodies recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed

Most RNA degradation occurs in P bodies recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed Also where AGO & miRNAs accumulate

Most RNA degradation occurs in P bodies recently identified cytoplasmic sites where exosomes & XRN1 accumulate when cells are stressed Also where AGO & miRNAs accumulate w/o miRNA P bodies dissolve!

Other ncRNA? Incredible diversity of functions! Epigenetic Directly regulating transcription Post-transcriptional regulation

Other ncRNA? Incredible diversity of functions! Epigenetic Directly regulating transcription Post-transcriptional regulation Some are made by Pol II, others by Pol III

Other ncRNA? Incredible diversity of functions!

Other ncRNA? Epigenetic regulation: guide &scaffold for chromatin mods XIST in mammals controls X-inactivation Transcribed by Pol II only from the inactive X 17 kb, -> Spliced, capped, poly-A Coats the inactive X Recruits PRC2, which silences the chromosome Ubiquitinates H2AK119 Tri-methylates H3K27 Hypermethylates H3K9

Other ncRNA? Epigenetic regulation: guide &scaffold for chromatin mods FLC blocks flowering in fall; after 20 days near 0˚C plants make COLDAIR ncRNA

FLC blocks flowering in fall; after 20 days near 0˚C plants make COLDAIR ncRNA: Targets PRC2 to FLC locus & silences it Flower next Spring Basic rule for epigentic ncRNA Bind target locus & recruit chromatin modifiers

ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator

ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock

ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced. The ncRNA acts as a coactivator for steroid hormone receptors

ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced.The ncRNA acts as a coactivator for steroid hormone receptors. The protein acts as a repressor of the ncRNA!

ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced.The ncRNA acts as a coactivator for steroid hormone receptors. The protein acts as a repressor of the ncRNA! ncRNA CCND1 are transcribed from the cyclin D locus during stress and remain tethered to DNA and recruit and allosterically modulate the protein TLS

ncRNA directly regulating transcription 1.Gene-specific Activating ncRNAs bind DNA & recruit Mediator heat shock RNA1 (HSR1) mobilizes heat shock factor 1 (HSF1) upon heat shock The steroid receptor RNA activator 1 locus encodes both a protein and an ncRNA, depending on how it is spliced.The ncRNA acts as a coactivator for steroid hormone receptors. The protein acts as a repressor of the ncRNA! ncRNA CCND1 are transcribed from the cyclin D locus during stress and remain tethered to DNA and recruit and allosterically modulate the protein TLS Other ncRNAs remain bound to the DNA coding sequence & prevent transcription

ncRNA directly regulating transcription 1.Gene-specific Other ncRNAs remain bound to the DNA coding sequence & prevent transcription Many sense and antisense ncRNA are found around promoters

ncRNA directly regulating transcription 1.Gene-specific Other ncRNAs remain bound to the DNA coding sequence & prevent transcription Many sense and antisense ncRNA are found around promoters Most are rapidly degraded: defective?

ncRNA directly regulating transcription 1.Gene-specific Other ncRNAs remain bound to the DNA coding sequence & prevent transcription Many sense and antisense ncRNA are found around promoters Most are rapidly degraded: defective? Loops may help identify correct direction

ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core

ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core

ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core

ncRNA directly regulating transcription 1.Gene-specific Many enhancer elements are transcribed by Pol II, this somehow enhances transcription from the core Looping brings many related genes close, where [Pol II] is high

ncRNA directly regulating transcription 1.Gene-specific 2.General U1 snRNA associates with TFIIH to stimulate Pol II CTD phosphorylation hence transcription Couples splicing with transcription

ncRNA directly regulating transcription General U1 snRNA associates with TFIIH to stimulate Pol II CTD phosphorylation hence transcription Upon heat shock SINE (Alu) elements are transcribed by Pol III; Alu RNA binds Pol II and prevents initiation

ncRNA directly regulating transcription U1 snRNA associates with TFIIH to stimulate Pol II CTD phosphorylation hence transcription Upon heat shock SINE (Alu) elements are transcribed by Pol III; Alu RNA binds Pol II and prevents initiation P-TEFb (an elongation factor) reversibly associates with 7SK ncRNA to regulate Pol II elongation

ncRNA indirectly regulating transcription 1.Decoys For transcription factors: eg Gas5 RNA binds the DNA- binding domain of steroid hormone receptors NRON (noncoding repressor of NFAT) RNA prevents NFAT from shuttling into the nucleus

ncRNA regulating genes post-transcriptionally 1.Decoys for miRNAs (or siRNAs) Many mammalian pseudogenes, including PTENP1 and KRASP1 have miRNA binding sites

ncRNA regulating genes post-transcriptionally 1.Decoys for miRNAs (or siRNAs) Many mammalian pseudogenes, including PTENP1 and KRASP1 have miRNA binding sites 2.Thousands of genes are transcribed in both sense and antisense directions

Thousands of antisense transcripts in plants 1.Overlapping genes

Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs

Thousands of antisense transcripts in plants 1.Overlapping genes 2.Non-coding RNAs 3.cDNA pairs 4.MPSS 5.TARs

Thousands of antisense transcripts in plants Hypotheses 1.Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al)

Hypotheses 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) 2. Functional a.siRNA b.miRNA c.Silencing

Hypotheses 1. Accident: transcription unveils “cryptic promoters” on opposite strand (Zilberman et al) 2. Functional a.siRNA b.miRNA c.Silencing d.Priming: chromatin remodeling requires transcription!

Detecting ncRNA 1. cDNA pairs

Detecting ncRNA 1.cDNA pairs 2.MPSS

Detecting ncRNA 1.cDNA pairs 2.MPSS 3.Tiling path microarrays

Detecting ncRNA 1.cDNA pairs 2.MPSS 3.Tiling path microarrays 4.RNA-seq (high-throughput RNA sequencing)

Our ncRNA 1.Extracted total RNA, separated out and sequenced nt

Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc)

Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find mutants

Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find genes that weren’t near protein- coding genes

Our ncRNA 1.Extracted total RNA, separated out and sequenced nt 2.BLAST to find relatives (and discard tRNA, etc) 3.T-DNA express to find genes that weren’t near protein- coding genes 4.33 Hits: order seeds