Fabian Lectures Flashcards
What are Cis-Elements?
Found in RNA & mediate interactions with specific proteins or ncRNA.
Often found as a short sequence.
Sometimes found as a folded sequence (ie., Stem-loop, hairpin)
What are Trans-Acting Factors?
RNA Binding Proteins (RBPs) or ncRNA (miRNA).
Binds to cis-acting sequences to control gene expression.
What are various elements of postranscriptional regulation?
M7G Cap
5’ UTR
3’ UTR
Open Reading Frame
3’ Poly (A) Tail
What are key post-transcriptional regulation features in Histone mRNAs?
Histone stem loop
1) SLBP = stem loop binding protein
2) SLIP = SLBP interacting protein
How does the 5’ cap and 3’ Poly(A) tail play critical roles in regulating mRNA translation and stability?
1) EIF4E binds to 5’ cap to stimulate mRNA translation.
2) Cytoplasmic Poly(A) binding protein, PABPC binds to mRNA 3’ poly (A) tail.
3) mRNA termini can interface via EIF4G, binding to EIF4E and PABP to stimulate mRNA translation.
How do trans-acting modulate the expression of its target?
It must associate with specific cis-elements within the mRNA.
What are the 3 cis-elements? How are they classified and organized?
1) AREs: AU-rich elements.
2) PRE: Pumilio response elements.
3) Speed elements: miRNA binding sites.
- they are classified into categories based on their sequence and function of structure.
- cis-elements can be organzied into consensus motifs recognized by trans-acting factors.
What are two types of trans-acting factors and what processes do they play roles in?
1) RNA-Binding Proteins (RBPs).
- highly abundant family of proteins (>2000 in eukaryotes)
- contain one or more RNA binding domains through which they bind to specific cis-elements located in 5’ or 3’ untranslated regions (UTRs) of distinct subpopulations of RNAs.
- effects processing & QC control, mRNA export, turnover, translation and transcription.
2) ncRNAs (miRNAs).
- family of non-coding single-stranded RNAs.
- bind to specific sequences, called ‘seed elements’ within the 3’ UTR to block translation, and promote decay of target mRNAs (some cases).
- effects turnover and translation.
What does post-transcriptional dys-regulation lead to?
- cancer
- inflammatory diseases
- muscular dystrophy
- autism
- parkinsons
- amyotrophic lateral sclerosis
What is a strategy to identify cis-acting motifs for RNA binding proteins?
1) RNACompete
- Use epitope tagged RBP (eg., GST) in a GST pulldown assay and bind complex RNA pool. The bound RNAs are eluted, directly labeled, and analyzed through microarray hybridization to determine the RNA motifs recognized by the RBP.
How do we identify the RNA targets of an RNA binding protein?
—> RIP-Immunoprecipitation Sequencing (RIP-Seq)
- Identifies the mRNA targets of an RNA-binding protein.
1) Cell Lysis (Lysis buffer)
2) IP the RBP (antibody coupled to magnetic bead)
3) Isolate mRNAs (dissociation step)
4) Identify mRNAs (sequencing)
5) Validate
–> Antibody, antibody-RBP target run through gel.
—> Reverse transcription, qPCR, primers for specific mRNA.
What are advantages and disadvantages of RIP-Seq?
Advantages:
1) use routine and regular methods such as immunoprecipitation and cell culture.
2) identifies the target mRNAs for various RBPs.
3) can be adapted to use recombination protein instead of an antibody,
Disadvantages:
1) limited to the characterization of kinetically stable interactions and is a snapshot of interactions.
2) does not identify the RBP binding site in the mRNA.
3) False positive interactions may occur upon lysis.
What is a method to identify the RNA-binding protein binding sites on an mRNA?
–> Crossline and Immunoprecipiation (CLIP)
1) Preparation of cross-linked cell lysate.
- covalent protein RNA-cross linking, cell lysis, RNA fragmentation.
2) Purification of specific cross-linked RNA fragments.
- Immunoprecipiation or affinity purification of protein-RNA complexes, ligation of adapter to fragmented RNA, purification of protein-bound RNA by SDS-page.
3) cDNA library sequencing and analysis.
- Reverse transcription, PCR & high-throughput sequencing, bioinformatics determination of binding peaks, integration with complementary data. Or validate with gel shifts and reporter constructs.
What is mRNA turnover?
- encompasses all the elements of mRNA stability and decay mechanisms.
-defined as the length of time an mRNA molecule is stable inside the cell. - the majority of mRNAs are unstable due to the presence of destabilizing cis-elements (Vs. housekeeping genes).
- these cis-elements are protected by stabilizing factors called RNA-binding proteins (RBPs).
- both cis and trans acting factors impact mRNA half lives. Both are involved in the process of decay by accelerating or slowing down the rate of deadenyaltion and cap removal.
- modulation of mRNA turnover by cis- (3’ UUTR) & trans-acting factors (RBPs).
destabilizing RBPs = short mRNA 1/2 life
stabilizing RBPs = medium mRNA 1/2 life
two stabilizing RBPs = long mRNA 1/2 life
What are the steps of cytoplasmic mRNA decay and the key factors involved in this process?
1) Deadenylation.
- before mRNA can be degraded, poly(A) tail removed by PAN2-PAN3 and/or CCR4-NOT deadenylation complexes.
- this releases PABPC & weakens interaction of eIF4E with 5’ cap.
2) Decapping.
- Decapping machinery access and remove 5’ cap.
- LSM1-7 complex associate with oligo(A) tails or 3’ uridyl tails to help recruit decapping machinery.
3) Decay.
- Degradation of the mRNA in the 5’ to 3’ direction by 5’ 3’ exoribonuclease 1 (XRN1) or in 3’-5’ direction by cytoplasmic exosome complex.
What is the biphasic deadenylation mechanism? (in cytoplasmic mRNA decay)
- CCR4 is a general deadenylase, which can degrade poly(A) RNA when bound by PABPC.
- CAF1 (CRR4-associated factor 1) is a specialized deadenylase that degrades naked poly(A) RNA and is blocked by PABP.
Uridylation can mark mRNAs for degradation. What enzyme does this?
- TUTases:
TUT4 & TUT7 selectively uridylate deadenylated mRNAs with short (>25 nt) poly(A) tails and promotes decay.
Deadenylation-independent decay (cytoplasmic mRNA decay)
Unlike exoribonucleases, endoribonucleases can cleave RNA transcripts at internal positions, giving rise to fragments which can then be targeted by 5’ to 3’ and 3’ to 5’ exoribonucleases.
How is the half-life of mRNA determined?
- the half-life of an mRNA is defined as the time at which 50% of the message remains upon inhibition of pol-II mediated transcription.
- this technique measures the decay of mRNA over time after transcription inhibition.
- chemicals can be used to achieve this (inhibit transcription)
What method can assess mRNA turnover?
1) Northern blot of ACT1 mRNA.
- Shut off transcription
- Isolate mRNA at several time points after transcriptional shut-down.
- determine the % of mRNA remaining at each time point after transcriptional shut-off.
- Graph level of RNA remaining for each time point on a semi-log graph.
slope = rate of mRNA decay.
time a which half of mRNA is degraded is the half life of mRNA.
2) Can also determine deadenylation rates experimentally using RNAaseH Northern Blot.
How can you Assess mRNA metabolism?
- Use metabolic labeling and Sequencing:
SLAM-Seq.
Thiol(SH)-linked alkylation for metabolic sequencing of RNA.
How is the ‘CCR4-NOT’ recruited for select mRNAs?
(complex used to deadenylate)
- interacts with RBPs.
- regulation by RBPs.
–> TTP (trans-acting factor which binds to cis-element) - ARE: AU-Rich element.
- PRE: Pumilio regulatory element.
What is a brief description of the steps involved in micro-RNA mediated gene silencing?
1) Transcriptional repression
- EIF4A, EIF4E, EIF4G
2) mRNA deadenylation
- CCR4-NOT
- GW182
- PAN3-PAN2
- PABP
-AGO on miRNA binding site
3) mRNA decapping
- DCP1, DCP2
4) 5’ - 3’ mRNA decapping
- XRN1
(all is working upwards to inhibit transcription)
AU-Rich elements (AREs)
- cis-element
- found in the 3’ untranslated region (3’ UTR) of many mRNAs that encode proto-oncogenes, nuclear transcription factors and cytokines.
- AREs (AUUA) are one of the most common determinants of RNA stability in mammalian cells.
TTP (Tristetraprolin, aka ZFP36)
- trans-acting factor
- an RBP that binds directly to AREs in select mRNA 3’ UTRs in order to recruit the CCR4-NOT complex.
- in between it TTP/ARE and CCR4-NOT complex is IDR (intrinsically disordered region, region of a protein that lacks fixed or ordered 3D structure).
Pumilio Proteins
- cis-element
- regulate stem cell fate, development and neurological functions.
- dysfunction of pumilio proteins contributes to neurodegeneration, epilepsy, infertility and cancer.
Roquin-mediated mRNA decay
- constitute decay element (CDE) represent a class or conserved RNA stem loops.
- roquin binds to CDE stem loop (high specificity)
- roquin causes mRNA deadenylation by recruitment of Ccr4-Caf1-NOT complex.
- in macrophages, roquin limites TNF-alpha production via CDE-mediated mRNA decay.
- regulators of inflammation, immunity, development.
How can you modify the message of RNA?
- m6A
- ‘writer’ = WTAP, METT:3, METTLI4. (nucleus)
- ‘reader’ = YTH, eIF3 (nucleus and cytosol)
- ‘erase’ = ALKBH5 (nucleus)
1) mRNA vaccines: can modify nucleosides, purified mRNA.
existence of methylated mRNA in mouse L cells.
2) RNA epigenetic: reversible DNA methylation, reversible RNA methylation, reversible histone methylation or acetylation. = all chemical modifications.
3) isolating methylated RNA:
- fragmentation to around 100 nucleotides.
- then immunoprecipiation with m6A - specific antibodies.
- input control.
- cDNA library construction and high-throughput sequencing, can find the location of m6A. it is mostly where the stop-codon is.
How can recruitment of CCR4-NOT to select mRNAs be regulated?
- RBP competition to regulate mRNA stability.
(A)
- Destabilizing RBPs:
AUF1
ZPF3oL 1/2
TTP
- stabilizing RBP
HuR
both bind to AU-rich element.
TTP gets phosphorylated by PP2A leading to mRNA decay and dephosphorylated leading to PP2A to mRNA stability.
(B)
- Destabilizing RBPs:
Regnase -1/3
Roquin 1/2
- stabilizing RBP
ARID5A
both bind to stem loop.
Regnase-1/3 gets phosphorylated by IRAKs, TBKI, IKKi and that leads to decay.
Regulation of PUM-mediated mRNA decay
- PUM = pumilio = cis-acting elements
- NORAD is broadly expressed, highly abundant, and conserved mammalian noncoding RNA (lncRNA).
- NORAD expression is up regulated under conditions of DNA damage and functions as a potent molecular decoy for Pumilio proteins (PUM1/PUM2)
- PUM1/PUM repress a program of genes necessary to maintain genomic stability.
- when DNA = damaged, NORAD protects the genes needed for DNA repair by blocking PUM1/PUM2 proteins.
- when NORAD +/+, normal pumilio activity and normal mitosis.
- when NORAD -/-, hyperactive pumiio and abberant mitosis.
How can we change miRNA levels?
- altered expression.
- altered miRNA biogenesis.
How can we make changes in mRNA target?
- chromosomal translocations.
- alternative splicing (modified 3’ UTRs - 3’ UTR shortening)
- alternative polyadenylation
Dicer1 syndrome & alterations in microRNA populations.
Dicer1 = gene that encodes Dicer protein. (essential for processing precursor miRNAs to mature miRNAs)
Germ line mutations (inherited mutations) in one copy of Dicer1 gene. A somatic “second hit” in cells can lead to complete loss of Dicer function in those cells.
At risk for developing many tumours.
Codon optimality and mRNA stability
- ability of a given codon to affect mRNA stability in a translation-dependent manner.
- distinct from codon usage.
- synonymous codon substitutions based on codon optimality predictions can lead to >30-fold difference in mRNA half life.
- downstream effects of codon optimality: mRNA stability, mRNA level, Poly-A-tail, translational efficiency, protein level.
Codon optimality-mediated mRNA decay (COMD)
1) Ribosome speed dictates transcript stability via general mRNA decay pathway.
2) Slow ribosome movement is sensed by deadenylase complex.
3) Ribosome speed is determined by codon optimality, which is a function of tRNA concentration.
slow = unstable mRNA.
fast = stable mRNA.
Codon optimality and mRNA stability in Yeast
- In yeast, CCR4-NOT can interact with ribosome, with one subunit of this complex binding to Ribosome E-site, stimulating deadenylation and decapping.
While the impact of COMD is a major determinant of mRNA half-lives in yeast, its impact on human mRNA stability is less clear.
What is a type of mRNA quality control?
Non-sense mediated decay (NMD)
1) NMD activation.
2) Substrate degredation.
What are Hallmarks of NMD?
- translation-dependent.
- degraded aberrant mRNAs: that include premature termination codons, abberant splicing or errors.
- degrades functional mRNAs: have introns in their 3’ UTR or 5’ UORFs.
- mediated by specific and general RNases.
- variable activity.
- affect genetic & neuronal diseases.
- target some viral mRNAs.
- enhances by EJC’s: exon-junction complexes downstream of termination codon stimulate nMD.
- mediated by specific and general RNAses.
What are some things that NMD activity can affect?
- the presentation of neoantigens at surface of cancer cells.
1) NMD can prevent or reduce pathological impact of some mutations in tumour suppressor genes by preventing translation.
2) will often be involved in reducing the effects of mutations that generate indels (insertions/deletions)
3) may also prevent cancer neoantigen production, may be targets of immune response. - cancer cells with low NMD activity: peptides from aberrant mRNAs presented by MHC Class I - immune response stimulation.
- cancer cells with high NMD activity: depletes cancer cell-specific antigen presentation cancer cells persist.
What is the connection between NMD and neuronal development and neurodegenerative diseases?
1) Point mutations and copy number variations in genes encoding NMD factors are associated with intellectual disability, including schizophrenia, ASD, ADHD.
2) UP53D KO in mice shows defects in neural stem cell differentiation.