BIO 369 - Resource and Policy Information Instructor: Dr. William Terzaghi Office: SLC 363/CSC228 Office hours: MW 11-12 and T 1-2 in SLC 363, R 1-2 & F 11-12 in CSC228, or by appointment Phone: (570) 408-4762 Email: terzaghi@wilkes.edu Course webpage: http://staffweb.wilkes.edu/william.terzaghi/bio369.html

Topics? Trying to find another way to remove oxalate Making a probiotic bacterium that removes oxalate Engineering magnetosomes to express novel proteins Studying ncRNA Studying sugar signaling Bioremediation Making plants/algae that bypass Rubisco to fix CO2 Making novel biofuels Making vectors for Dr. Harms Something else?

Genome Projects Studying structure & function of genomes Sequence first Then location and function of every part

Molecular cloning To identify the types of DNA sequences found within each class they must be cloned Force host to make millions of copies of a specific sequence How? 1) create recombinant DNA 2) transform recombinant molecules into suitable host 3) identify hosts which have taken up your recombinant molecules 4) Extract DNA

Molecular cloning usually no way to pick which fragment to clone solution: clone them all, then identify the clone which contains your sequence construct a library, then screen it to find your clone a collection of clones representing the entire complement of sequences of interest 1) entire genome for genomic libraries 2) all mRNA for cDNA

Libraries Why? Genomes are too large to deal with: break into manageable “volumes”

Libraries How? randomly break DNA into vector-sized pieces & ligate into vector 1) partial digestion with restriction enzymes 2) Mechanical shearing

reverse transcriptase makes DNA copies of all mRNA molecules present Libraries How? B) make cDNA from mRNA reverse transcriptase makes DNA copies of all mRNA molecules present mRNA can’t be cloned, DNA can

All the volumes of the library look the same Detecting your clone All the volumes of the library look the same trick is figuring out what's inside usually done by “screening” the library with a suitable probe identifies clones containing the desired sequence

Detecting your clone Probes = molecules which specifically bind to your clone Usually use nucleic acids homologous to your desired clone Sequences cloned from related organisms or made by PCR Make them radioactive, fluorescent, or “tagged” some other way so they can be detected

Detecting your clone by membrane hybridization Denature Transfer to a filter 3) probe with complementary labeled sequences 4) Detect radioactivity -> detect by autoradiography biotin -> detect enzymatically

Analyzing your clone 1) FISH 2) “Restriction mapping” a) determine sizes of fragments obtained with different enzymes b) “map” relative positions by double digestions

Southern analysis 1) digest genomic DNA with restriction enzymes 2) separate fragments using gel electrophoresis 3) transfer & fix to a membrane 4) probe with your clone

Northern analysis 1) fractionate by size using gel electrophoresis 2) transfer & fix to a membrane 3) probe with your clone 4) determine # & sizes of detected bands tells which tissues or conditions it is expressed in intensity tells how abundant it is

RT-PCR First reverse-transcribe RNA, then amplify by PCR Can make cDNA of all RNA using poly-T and/or random hexamer primers

RT-PCR First reverse-transcribe RNA, then amplify by PCR Can make cDNA of all RNA using poly-T and/or random hexamer primers Can do the reverse transcription with gene-specific primers.

Quantitative (real-time) RT-PCR First reverse-transcribe RNA, then amplify by PCR Measure number of cycles to cross threshold. Fewer cycles = more starting copies

Quantitative (real-time) RT-PCR First reverse-transcribe RNA, then amplify by PCR Measure number of cycles to cross threshold. Fewer cycles = more starting copies Detect using fluorescent probes

Quantitative (real-time) RT-PCR Detect using fluorescent probes Sybr green detects dsDNA

Quantitative (real-time) RT-PCR Detect using fluorescent probes Sybr green detects dsDNA Others, such as taqman, are gene-specific

Quantitative (real-time) RT-PCR Detect using fluorescent probes Sybr green detects dsDNA Others, such as taqman, are gene-specific Can multiplex by making gene-specific probes different colors

Western analysis Separate Proteins by PAGE 2) transfer & fix to a membrane

Western analysis 1) Separate Proteins by polyacrylamide gel electrophoresis 2) transfer & fix to a membrane 3) probe with suitable antibody (or other probe) 4) determine # & sizes of detected bands

Analyzing your clone 1) FISH 2) “Restriction mapping” 3) Southern analysis : DNA 4) Northern analysis: RNA 5) Sequencing

DNA Sequencing Basic approach: create DNA molecules which start at fixed location and randomly end at known bases

DNA Sequencing Basic approach: create DNA molecules which start at fixed location and randomly end at known bases generates set of nested fragments

DNA Sequencing Basic approach: create DNA molecules which start at fixed location and randomly end at known bases generates set of nested fragments separate these fragments on gels which resolve molecules differing in length by one base

DNA Sequencing Basic approach: create DNA molecules which start at fixed location and randomly end at known bases generates set of nested fragments separate these fragments on gels which resolve molecules differing in length by one base creates a ladder where each rung is 1 base longer than the one below

DNA Sequencing Basic approach: create DNA molecules which start at fixed location and randomly end at known bases generates set of nested fragments separate these fragments on gels which resolve molecules differing in length by one base creates a ladder where each rung is 1 base longer than the one below read sequence by climbing the ladder

DNA Sequencing Sanger (di-deoxy chain termination) 1) anneal primer to template

DNA Sequencing Sanger (di-deoxy chain termination) 1) anneal primer to template 2) elongate with DNA polymerase

DNA Sequencing Sanger (di-deoxy chain termination) 1) anneal primer to template 2) elongate with DNA polymerase 3) cause chain termination with di-deoxy nucleotides

DNA Sequencing Sanger (di-deoxy chain termination) 1) anneal primer to template 2) elongate with DNA polymerase 3) cause chain termination with di-deoxy nucleotides will be incorporated but cannot be elongated 4 separate reactions: A, C, G, T

DNA Sequencing Sanger (di-deoxy chain termination) 1) anneal primer to template 2) elongate using DNA polymerase 3) cause chain termination with di-deoxy nucleotides 4) separate by size Read sequence by climbing the ladder

Automated DNA Sequencing 1) Use Sanger technique 2) label primers with fluorescent dyes Primer for each base is a different color! A CGT 3) Load reactions in one lane 4) machine detects with laser & records order of elution

Genome projects 1) Prepare map of genome

Genome projects Prepare map of genome To find genes must know their location

Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library FISH to find their chromosome

Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library FISH to find their chromosome identify overlapping AC using ends as probes assemble contigs until chromosome is covered

Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library 4) Subdivide each AC into lambda contigs

Sequencing Genomes 1) Map the genome 2) Prepare an AC library 3) Order the library 4) Subdivide each AC into lambda contigs 5) Subdivide each lambda into plasmids 6) sequence the plasmids

Using the genome Studying expression of all genes simultaneously Microarrays (reverse Northerns) Attach probes that detect genes to solid support

Using the genome Studying expression of all genes simultaneously Microarrays (reverse Northerns) Attach probes that detect genes to solid support cDNA or oligonucleotides

Using the genome Studying expression of all genes simultaneously Microarrays (reverse Northerns) Attach probes that detect genes to solid support cDNA or oligonucleotides Tiling path = probes for entire genome

Microarrays (reverse Northerns) Attach probes that detect genes to solid support cDNA or oligonucleotides Tiling path = probes for entire genome Hybridize with labeled targets

Microarrays Attach cloned genes to solid support Hybridize with labeled targets Measure amount of target bound to each probe

Microarrays Measure amount of probe bound to each clone Use fluorescent dye : can quantitate light emitted

Microarrays Compare amounts of mRNA in different tissues or treatments by labeling each “target” with a different dye

Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” Fix probes to slide at known locations, hyb with labeled targets, then analyze data

Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” High-throughput sequencing

Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” High-throughput sequencing “Re-sequencing” to detect variation

Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” High-throughput sequencing “Re-sequencing” to detect variation Sequencing all mRNA to quantitate gene expression

Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” High-throughput sequencing “Re-sequencing” to detect variation Sequencing all mRNA to quantitate gene expression Sequencing all mRNA to identify and quantitate splicing variants

Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” High-throughput sequencing “Re-sequencing” to detect variation Sequencing all mRNA to quantitate gene expression Sequencing all mRNA to identify and quantitate splicing variants Sequencing all RNA to identify and quantitate ncRNA

Using the genome Studying expression of all genes simultaneously Microarrays: “reverse Northerns” High-throughput sequencing Bisulfite sequencing to detect C methylation

Using the genome Bisulfite sequencing to detect C methylation

Using the genome Bisulfite sequencing to detect C methylation ChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells

Using the genome various chromatin modifications are associated with activated & repressed genes Acetylation, egH3K9Ac, is associated with active genes

Using the Genome various chromatin modifications are associated with activated & repressed genes Acetylation, egH3K9Ac, is associated with active genes Phosphorylation of H2aS1, H2aT119, H3T3, H3S10 & H3S28 shows condensation

Using the Genome Acetylation, egH3K9Ac, is associated with active genes Phosphorylation shows condensation Ubiquitination of H2A and H2B shows repression & marks DNA damage

Using the Genome Acetylation, egH3K9Ac, is associated with active genes Phosphorylation shows condensation Ubiquitination of H2A and H2B shows repression Methylation is more complex: H3K36me3 = on H3K27me3 = off

Using the Genome Methylation is more complex: H3K36me3 = on H3K27me3 = off H3K4me1 = off H3K4me2 = primed H3K4me3 = on

Histone code Modifications tend to group together: genes with H3K4me3 also have H3K9ac

Histone code Modifications tend to group together: genes with H3K4me3 also have H3K9ac Cytosine methylation is also associated with repressed genes

Generating the histone code Histone acetyltransferases add acetic acid

Generating the histone code Histone acetyltransferases add acetic acid Many HAT proteins: mutants are very sick!

Generating the histone code Histone acetyltransferases add acetic acid Many HAT proteins: mutants are very sick! HATs are part of many complexes

Generating the histone code Bromodomains specifically bind acetylated lysines

Generating the histone code Bromodomains specifically bind acetylated lysines Found in transcriptional activators & general TFs

Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate

Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate SIRT1 is activated by resveratrol

Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate SIRT1 is activated by resveratrol SIRT6 increases lifespan of male mice

Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate Deacetylase mutants are sick!

Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors

Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors SAHA = suberanilohydroxamic acid = vorinostat Merck calls it Zolinza, treats cutaneous T cell lymphoma

Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors SAHA = suberanilohydroxamic acid = vorinostat Merck calls it Zolinza, treats cutaneous T cell lymphoma Binds HDAC active site & chelates Zn2+

Generating the histone code When coupled SAHA to PIPS (pyrrole-imidazole Polyamides) got gene- specific DNA binding & gene activation

Generating the histone code CDK8 kinases histones to repress transcription

Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription

Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription Phosphorylation of Histone H3 correlates with activation of heat shock genes!

Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription Phosphorylation of Histone H3 correlates with activation of heat shock genes! Phosphatases reset the genes

Generating the histone code Acetylation, egH3K9Ac, is associated with active genes Phosphorylation shows condensation Ubiquitination of H2A and H2B shows repression & marks DNA damage

Generating the histone code Ubiquitination of H2A and H2B shows repression & marks DNA damage Rad6 proteins ubiquitinate histone H2B to repress transcription

Using the Genome Rad6 proteins ubiquitinate histone H2B to repress transcription Other proteins ubiquitinate H2B to enhance transcription!

Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription: others enhance it! Polycomb proteins ubiquitinate histone H2A to silence genes

Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription: others enhance it! Polycomb proteins ubiquitinate H2A to silence genes Multiple de-ubiquitinases have been identified

Generating the histone code Many proteins methylate histones: highly regulated!

Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity

Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy!

Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy! Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)

Generating the histone code Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red) Histone demethylases have been recently discovered

Generating methylated DNA Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE

Generating methylated DNA Si RNA are key: RNA Pol IV generates antisense or foldback RNA, often from TE RDR2 makes it DS, 24 nt siRNA are generated by DCL3

Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V

Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V Pol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target site

Generating methylated DNA RDR2 makes it DS, 24 nt siRNA are generated by DCL3 AGO4 binds siRNA, complex binds target & Pol V Pol V makes intergenic RNA, associates with AGO4-siRNA to recruit “silencing Complex” to target site Amplifies signal! extends meth- ylated region

Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data

Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data Many different sites provide “digital Northerns” or other comparative analyses of gene expression http://cgap.nci.nih.gov/SAGE http://www.weigelworld.org/research/projects/geneexpressionatlas

Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data Many different sites provide “digital Northerns” or other comparative analyses of gene expression http://cgap.nci.nih.gov/SAGE http://www.weigelworld.org/research/projects/geneexpressionatlas MPSS (massively-parallel signature sequencing) http://mpss.udel.edu/

Using the genome Many sites provide gene expression data online NIH Gene expression omnibus http://www.ncbi.nlm.nih.gov/geo/ provides access to many different types of gene expression data Many different sites provide “digital Northerns” or other comparative analyses of gene expression http://cgap.nci.nih.gov/SAGE http://www.weigelworld.org/research/projects/geneexpressionatlas MPSS (massively-parallel signature sequencing) http://mpss.udel.edu/ Use it to decide which tissues to extract our RNA from

Using the genome Many sites provide gene expression data online Many sites provide other kinds of genomic data online http://encodeproject.org/ENCODE/

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

Post-transcriptional regulation Nearly ½ of human genome is transcribed, only 1% is coding 98% of RNA made is non-coding Fraction increases with organism’s complexity

Known NcRNAs classes and functions

Implication in diseases

Implication in diseases

Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (a1aIIbb’w)

Transcription Prokaryotes have one RNA polymerase makes all RNA core polymerase = complex of 5 subunits (a1aIIbb’w) w not absolutely needed, but cells lacking w are very sick

Initiating transcription in Prokaryotes 1) Core RNA polymerase is promiscuous

Initiating transcription in Prokaryotes Core RNA polymerase is promiscuous sigma factors provide specificity

Initiating transcription in Prokaryotes Core RNA polymerase is promiscuous sigma factors provide specificity Bind promoters

Initiating transcription in Prokaryotes Core RNA polymerase is promiscuous sigma factors provide specificity Bind promoters Different sigmas bind different promoters

Initiating transcription in Prokaryotes Core RNA polymerase is promiscuous sigma factors provide specificity Bind promoters 3) Once bound, RNA polymerase “melts” the DNA

Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template

Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template

Initiating transcription in Prokaryotes 3) Once bound, RNA polymerase “melts” the DNA 4) rNTPs bind template 5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template 6) sigma falls off after ~10 bases are added

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by s factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by s factor 2)” -35 region” : 5’-TTGACA-3’ : bound by s factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by s factor 2)” -35 region” : 5’-TTGACA-3’ : bound by s factor 3) UP element : -57: bound by a factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by s factor 2)” -35 region” : 5’-TTGACA-3’ : bound by s factor 3) UP element : -57: bound by a factor

Structure of Prokaryotic promoters Three DNA sequences (core regions) 1) Pribnow box at -10 (10 bp 5’ to transcription start) 5’-TATAAT-3’ determines exact start site: bound by s factor 2)” -35 region” : 5’-TTGACA-3’ : bound by s factor 3) UP element : -57: bound by a factor Other sequences also often influence transcription! Eg Trp operator

Prok gene regulation 5 genes (trp operon) encode trp enzymes

Prok gene regulation Copy genes when no trp Repressor stops operon if [trp]

Prok gene regulation Repressor stops operon if [trp] trp allosterically regulates repressor can't bind operator until 2 trp bind

lac operon Some operons use combined “on” & “off” switches E.g. E. coli lac operon Encodes enzymes to use lactose lac Z = -galactosidase lac Y= lactose permease lac A = transacetylase

lac operon Make these enzymes only if: 1) - glucose

lac operon Make these enzymes only if: 1) - glucose 2) + lactose

lac operon Regulated by 2 proteins 1) CAP protein : senses [glucose]

lac operon Regulated by 2 proteins CAP protein : senses [glucose] lac repressor: senses [lactose]

lac operon Regulated by 2 proteins CAP protein : senses [glucose] lac repressor: senses [lactose] encoded by lac i gene Always on

lac operon 2 proteins = 2 binding sites 1) CAP site: promoter isn’t active until CAP binds

lac operon 2 proteins = 2 binding sites CAP site: promoter isn’t active until CAP binds Operator: repressor blocks transcription

lac operon Regulated by 2 proteins 1) CAP only binds if no glucose -> no activation

lac operon Regulated by 2 proteins 1) CAP only binds if no glucose -> no activation 2) Repressor blocks transcription if no lactose

lac operon Regulated by 2 proteins 1) CAP only binds if no glucose 2) Repressor blocks transcription if no lactose 3) Result: only make enzymes for using lactose if lactose is present and glucose is not

Result [-galactosidase] rapidly rises if no glucose & lactose is present W/in 10 minutes is 6% of total protein!

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter.

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. ↵

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. nrsBACD encode nickel transporters

Structure of Prokaryotic promoters Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. nrsBACD encode nickel transporters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator

Structure of Prokaryotic promoters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator When nrsS binds Ni it kinases nrsR

Structure of Prokaryotic promoters nrsRS encode “two component” signal transducers nrsS encodes a his kinase nrsR encodes a response regulator When nrsS binds Ni it kinases nrsR nrsR binds Ni promoter and activates transcription of both operons

Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind

Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho”

Termination of transcription in prokaryotes 1) Sometimes go until ribosomes fall too far behind 2) ~50% of E.coli genes require a termination factor called “rho” 3) rrnB first forms an RNA hairpin, followed by an 8 base sequence TATCTGTT that halts transcription

Transcription in Eukaryotes 3 RNA polymerases all are multi-subunit complexes 5 in common 3 very similar variable # unique ones Plants also have Pols IV & V make siRNA

Transcription in Eukaryotes RNA polymerase I: 13 subunits (5 + 3 + 5 unique) acts exclusively in nucleolus to make 45S-rRNA precursor

Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis

Transcription in Eukaryotes Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor accounts for 50% of total RNA synthesis insensitive to -aminitin

Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor 50 % of total RNA synthesis insensitive to -aminitin Mg2+ cofactor Regulated @ initiation frequency

Processing rRNA ~ 100 bases are methylated C/D box snoRNA pick sites One for each!

Processing rRNA ~ 100 bases are methylated C/D box snoRNA pick sites One for each! ~ 100 Us are changed to PseudoU H/ACA box snoRNA pick sites

Processing rRNA ~ 100 bases are methylated C/D box snoRNA pick sites ~ 100 Us are changed to PseudoU H/ACA box snoRNA pick sites 3) Some snoRNA direct modification of tRNA and snRNA

Processing rRNA ~ 200 bases are modified 2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products by ribozymes RNase MRP cuts between 18S & 5.8S U3, U8, U14, U22, snR10 and snR30 also guide cleavage

Processing rRNA ~ 200 bases are methylated 2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products 3) Ribosomes are assembled w/in nucleolus

RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA, scRNA, etc) >100 different kinds of ncRNA ~10% of all RNA synthesis Cofactor = Mn2+ cf Mg2+ sensitive to high [-aminitin]

Processing tRNA tRNA is trimmed 5’ end by RNAse P (1 RNA, 10 proteins)

Processing tRNA tRNA is trimmed Transcript is spliced Some tRNAs are assembled from 2 transcripts

transferase (no template) Processing tRNA tRNA is trimmed Transcript is spliced CCA is added to 3’ end By tRNA nucleotidyl transferase (no template) tRNA +CTP -> tRNA-C + PPi tRNA-C +CTP--> tRNA-C-C + PPi tRNA-C-C +ATP -> tRNA-C-C-A + PPi

Processing tRNA tRNA is trimmed Transcript is spliced CCA is added to 3’ end Many bases are modified Protects tRNA Tweaks protein synthesis

Processing tRNA tRNA is trimmed Transcript is spliced CCA is added to 3’ end Many bases are modified No cap! -> 5’ P (due to 5’ RNAse P cut)

Splicing: the spliceosome cycle 1) U1 snRNP (RNA/protein complex) binds 5’ splice site

Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint

Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint 3) U4/U5/U6 complex binds intron displace U1 spliceosome has now assembled

Splicing: RNA is cut at 5’ splice site cut end is trans-esterified to branchpoint A

Splicing: 5) RNA is cut at 3’ splice site 6) 5’ end of exon 2 is ligated to 3’ end of exon 1 7) everything disassembles -> “lariat intron” is degraded

Splicing:The spliceosome cycle

Splicing: Some RNAs can self-splice! role of snRNPs is to increase rate! Why splice?

Splicing: Why splice? 1) Generate diversity exons often encode protein domains

Splicing: Why splice? 1) Generate diversity exons often encode protein domains Introns = larger target for insertions, recombination

Why splice? 1) Generate diversity >94% of human genes show alternate splicing

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant stress-response proteins

Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant Stress-response proteins Splice-regulator proteins control AS: regulated by cell-specific expression and phosphorylation

Why splice? Generate diversity Trabzuni D, et al (2013)Nat Commun. 22:2771. Found 448 genes that were expressed differently by gender in human brains (2.6% of all genes expressed in the CNS). All major brain regions showed some gender variation, and 85% of these variations were due to RNA splicing differences

Why splice? Generate diversity Wilson LOW, Spriggs A, Taylor JM, Fahrer AM. (2014). A novel splicing outcome reveals more than 2000 new mammalian protein isoforms. Bioinformatics 30: 151-156 Splicing created a frameshift, so was annotated as “nonsense-mediated decay” an alternate start codon rescued the protein, which was expressed

Why splice? Splicing created a frameshift, so was annotated as “nonsense-mediated decay” an alternate start codon rescued the protein, which was expressed Found 1849 human & 733 mouse mRNA that could encode alternate protein isoforms the same way So far 64 have been validated by mass spec

Regulatory ncRNA SiRNA direct DNA-methylation via RNA-dependent DNA-methyltansferase In other cases direct RNA degradation

mRNA degradation lifespan varies 100x Sometimes due to AU-rich 3' UTR sequences Defective mRNA may be targeted by NMD, NSD, NGD Other RNA are targeted by small interfering RNA

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into 21-28 bp

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into 21-28 bp helicase melts dsRNA

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into 21-28 bp helicase melts dsRNA - RNA binds RISC

Other mRNA are targeted by small interfering RNA defense against RNA viruses DICERs cut dsRNA into 21-28 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 21-28 bp helicase melts dsRNA - RNA binds RISC complex binds target target is cut

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