Study Guide Ch. 10 Gene Regulation and Epigenetics
What is a pathogenic synonymous mutation?
Change in DNA seq coding for amino acids in protein sequ, but doesn't change encoded amino acid IF transcript spliced at a cryptic splice site, it could alter amino sequence which could turn pathogenic.
Be able to give examples of cis- and trans-acting gene regulation. .
Cis-acting: UTR regions act on own transcript Ex.: Maria Unna Hypotrichosis (OMIM#146550) - HR (hairless) gene has 4 upstream ORFs in its 691 bp 5′ UTR → Loss-of-function mutations in inhib U2HR → overexpression of HR protein → Hair follicle cycling is disrupted → autosomal dominant hair-loss Trans-acting: Prod of remote RNAs act on separate RNA molecule A Ex.: microRNA (miRNA) - miRNA can affect translation of many targets. - Impede protein expression by binding & breaking down targeted mRNA
Have an understanding of CH Waddington's concept of an epigenetic landscape, interpreted in terms of super-enhancers and transcription factors.
Concept: "path of cell development is like a ball rolling down a series of hills & valleys towards a specific endpoint" Landscape → regulatory factors & epigenetic modifications Super-enhancers Transcription factors act in hirarchies small nr of master factors define cell identity by binding super enhancers
What are the different types of promoters elements and which type of promoter is used for expression of 70% of genes?
CpG Islands TATA Box 70% used in expression of Genes Initiator (Inr) helps position RNA poly Downstream promoter element (DPE) Motif ten element (MTE) Downstream core element (DCE) CAAT box enhance transcr → binding of TF GC box binding site for TF Enhancers Silencers Insulators
How does the outcome of C>U editing create different isoforms of the protein encoded by the APOB gene?
Liver: APOB encodes the large ApoB100 protein Intestine: C>U editing at nucleotide position 6666 of the mRNA causes replacement of the CAA glutamine codon by a UAA stop codon, which now encodes a shorter polypeptide, ApoB48.
Why might a mutation in enhancers and silencers result in disease?
Loss of function prevent activation or repression of transcription = reduced production of proteins ex.: beta-thalassemia, enhancer mutation = reduced beta-globin prod Gain of function new TF binding sites = increased gene expression = more protein produced ex.: mutation in enhancer insulin-like growth factor 2 (IGF2) gene = activation of cancer = promotes growth Alteration of specificity change binding specificity of reg elements = regulate wrong gene
What are some examples of "miRNA sponges"?
miRNA sponges → create loss-of-function phenotypes for miRNA families in cell culture & in virally infected tissue & transgenic animals Ex.: PTENP
How does the lack of A>I (QR) editing contribute to ALS (see slides)?
Motor neurons of sporadic ALS patients express unedited GluA2 mRNA at Q/R site in disease-specific & motor neuron-selective manner Normal: mediated by adenosine deaminase acting on mRNA (ADAR2) For AMPA receptors to be impermeable to Ca2+ the GluA2 must be Q/R site edited
Why is gene regulation considered to be more of a network than a one-dimensional process?
Multiple genes can interact with each other to regulate their expression Gene expression influenced by variety of internal/external factors & pathways → requires multiple levels of control Regulatory molecules → transcription factors & microRNAs can bind to & affect multiple genes at once → complex regulatory networks
Why would the transition of a paused RNA polymerase into elongation mode be important for gene regulation?
Provides mech for controlling timing & rate of transcription RNA poly inplace at promoter → allows rapid & reversible control of gene expr → RNA poly can quickly continue Paused RNA poly → subject to reg by other factors → ex.: chromatin remodelling → transition into elongation mode
Why might it be that some pseudogenes have undergone purifying selection.
Pseudogene - DNA segment → structurally resembles a gene → doesn't code for protein - been neglected → bad copies → lost coding potential → void of function Purifying Selection - removing alleles which are deleterious → reduces genetic diversity → at sites under direct selection & linked nautral sites - might consider pseudogenes → useless & deleterious
Where will the RNA polymerase bind? Where is the Ribosomal binding site?
RNA Polymerase RNA poly → binds promoter region → upstream of start codon RNA poly reads in 3' to 5' direction → synth RNA in 5' to 3' Ribosomal binding site located on mRNA → upstream of start codon → ribosome bind → initiate translation
What is RNA editing?
RNA editing involves the insertion, deletion, or modification of specific nucleotides in the primary transcript. Main events are deamination of cytosine or adenine.
What is a cryptic splice site?
Seq in pre-mRNA with resemblance to splice site, may be used as splice site when splicing is disturbed or after base substitution mutation that increases resemblance to normal splice site. Normally these sites are ignored by spliceosome, but potentially could act as splice sites for spliceosome selection.
What are the role of snRNPs in RNA splicing?
Small nuclear ribonucleoproteins (snRNPs) recognizing the critical sequence elements present in the introns, thereby forming active spliceosomes
What does it mean to have a strong or weak promoter?
Strong → high freq of transx Weak → low freq of transx
Be able to give examples of how transcript stability and translational rate are influenced by the 3' UTR and/or 5'UTR
Translational Regulation - 5'UTR contains key elements of trans reg → structural motifs & upstream open reading frames - controlling translation initiation sites selection → seq elements in 5'UTR contribute mRNA translatability - 3'UTR main site of miRNA binding & section of mRNA following translation termination codon → damages or instability → transcript stability to decrease
Know what alternative splicing is, its significance for creating different protein isoforms, how commonly it occurs and what controls it.
- Alt splicing allows 1 primary transcript to encode multi protein isoforms → exons from same gene are joined in diff combinations → diff, but related, mRNA transcripts - Splicing may be tissue-specific, so that diff tissues contain diff variants, - Spliced exons may encode signals governing diff intracellular localizations - >90% of human genes undergo some kind of alternative splicing - Many more proteins than genes - Increase functional variation
Be prepared to talk about DNA coding/template strand and how it relates to a final transcript with upstream and downstream regions to the actual coding sequence.
- Coding strand or sense strand, 5' to 3' → same seq as RNA transcript - Template strand or antisense, 3' to 5' → is transcribed - RNA transcript 3 regions: 5' UTR, coding region, 3' UTR - 3' UTR is downstream of coding region → contains reg seq - coding region starts with AUG start codon - ends with UAA, UAG or UGA
Would you consider RNA editing a normal healthy process, or does it lead to disease?
- Involved in various disorders inc cancer, and neurological diseases of brain or CNS. Related to cancer heterogeneity & onset of carcinogenesis - Intention is to create diversity, but it can lead to mutation that favors certain diseases Ex.: ALS and Epilepsy
What controls alternative splicing?
- Splice sites can be strong or weak → weak skipped in favor of alt - Splicing site often tissue specific → enhancers/suppressors bind tissue specific proteins - Epigenetic marks affect splicing → exons show epigen diff relative to introns → inc CpG methylation
Be able to list several different levels of regulation for protein production and function?
1) Transcriptional Regulation controlling the rate of transcription 2) Post-Transcriptional Regulation alter splicing of pre-mRNA or mRNA stability 3) RNA processing control RNA splicing, capping, & polyadenylation control 4) mRNA transport & localization control mRNA from nucleus to cytoplasm for translation 5) mRNA degradation control control mRNA stability → mRNA degradation 6) Translation control control protein prod → regulatory factors binding mRNA or ribosome 7) Post-translational regulation protein folding, modifications 8) Protein degradation control control rate of protein turnover → ubiquitin pathway 9) Protein activity control regulate protein func → modulation of protein-protein interactions, binding of allosteric effectors (change conformation)
Know each of the steps to processing pre-mRNA into mature RNA that were discussed in class and how they may play a role in protein production.
1. Addition of a 5' cap to beginning of RNA 5' cap → a 7-methylguanylate connected to tern 5'-5' triphosphate linkage Function: protect 5' end; aids nuclear export; assist translation in cytoplasm 2. Addition of poly-A tail (100-300 A) end of RNA Polynucleotide adenylyl transferase adds poly-A tail to 3' end Function: prevents degradation & assists export from nucleus 3. Splicing Introns are removed by spliceosome complex → consists of snRNAs + snRNPs a. nucleophilic attack intros at 5' by branch point A → lariat shaped structure b. cleavage of exon-intron junction c. nucleophilic attack at 3' end upstream of acceptor site → cleavage & release of intronic RNA as lariat → splicing together 2 exons
How are microRNA produced (very vaguely)?
1. Precursor miRNA formed in nucleus 2. RNA processing and export to cytoplasm 3. miRNA and RISC proteins assemble into RISC (RNA-induced silencing complex)
What is an interesting feature or the ORF(s) of the CDKN2A gene?
2 alternative 1st exons, and each contains a translational start site. Depending which is used → seq of shared downstream exons is translated into diff reading frames → same downstream exons encode totally diff proteins → depending on which promoter is used
What is DNA methylation, how and where does it occur and how does it influence gene expression? Does DNA methylation normally affect base-pairing? What happens when 5-meC undergoes spontaneous deamination?
Addition of methyl grp to C5 of Cytosine in CpG DNA methyltransferase catalyzes the process Methylation → gene expression is OFF → prevents TF binding Spontaneous deamination of 5-meC → leads to a C to T transition mutation
Why is A>I editing also called QR editing?
Adenosine (A) -> Inosine (I) Deamination of adenosine by ADAR (adenosine deaminase acting on RNA) Often the editing converts CAG codons, encoding glutamine (Q), into CIG codons that, like CGG, encode arginine (R).
What are alternative promoters?
Alternative promoters can involve different regulatory elements, such as allowing different regulation of gene expression in different tissues. They also allow functionally significant sequence differences between alternative first exons.
How many human genes contain more than one?
At least half of all genes use alternative promoters. 200,310 human transcripts 20,338 protein-coding genes.
Know the different types of DNA binding proteins that are important for gene expression and have a general understanding of where they bind relative to the promoter.
B (TFIIB) recognition element (BRE) Located upstream of promoter TFIIB Initiator Element (Inr) located downstream of promoter TFIID binds Downstream Promoter Element (DPE) downstream of promoter and further downstream then Inr TFIID
What are enhancers and silencers? How do they contribute to regulation?
Enhancers bind TF → increase rate of transcription initiation Silencers bind TF → decrease rate of transcription initiation Silencers + Enhancer contribute to reg by influencing recruitment of TF to promoter
Is hetero- or euchromatin more accessible to endonucleases? Why? Which would be more sensitive to DNases (nucleases that target DNA)?
Euchromatin is more accessible to endonucleases → bc less condensed & has a more open chromatin structure Heterochromatin more tightly packed- Euchromatin would also be more sensitive to DNases because of its more open chromatin structure
Could a gain or loss (or both) of poly adenylation activity be pathogenic? Why?
Gain of polyadenylation activity: addition of longer poly(A) tail → increase stability of mRNA → higher expression levels of protein pathogenic if protein is overproduced or if it interferes with normal cellular processes. Loss of polyadenylation activity: mRNA lacking a poly(A) tail → destabilizes mRNA → lower expression of protein pathogenic if protein is necessary for normal cellular processes or absence results in loss of func
What are upstream ORFs (uORF)? What role do they play in regulating protein production? (e.g. the HR (Hairless) gene)?
uORFs open reading frame → within 5'UTR of mRNA → can inhibit translation of main reading frame HR gene has 4 upstream ORFs in 691 bp 5'UTR → loss-function mutation → in inhibitory U2HR → HR protein overexpression → hair loss