Northern Blot
RNA rextraction + electrophoresis. Separate RNA by size and then transfer RNA
to membrane. Fix RNA with UV or heat to membrane and then hybridize with
labeled probes. Visualize label RNA on X-ray film. Can see gene expression
rates. [Western Blot]
Load and separate protein samples on SDS-Page and electrophoretically transfer
fractionated proteins into PVDF membrane. Block membrane with neutal
protein. Incubate the membrane with primary antibody specific to target protein
and then incubate with labeled secondary antibody specific to primary.Incubate
the bloth with chemiluminescent HRP substrate and expose to film. [PCR /
qPCR / RT-qPCR]
Rna isolation and analysis then first-strand cDNA synthesis:
Real time PCR: Real-time PCR amplification, continuous fluorescent
measurement of PCR product during each cycle of PCR. Then analysis of data.
Regular: standard PCR amplification, then DNA gel electrophoresis at
completion of PCR, quantification of visible PCR by densitometry, then analysis.
Method of detection by dyes with dsDNA or DNA probes with oligonucleotides
that are labeled with fluorescent reporter to certain sequence [CHIP Assay]
Cells fixed with formaldehyde to crosslink histone and non-histone proteins to
DNA. Then chromatin digested w/ nuclease into DNA/protein fragments.
Antibodies to specific histone of non-histone proteins added and complex coprecipitates and is captured by beads. Cross-links reversed and DNA ready for
analysis. Protein-DNA interactions. [CLIP] Crosslinking and
immunoprecipitation
Crosslink before you lyse the cell. Then covalent bond between RNAbp and
RNA, and that allows to do a more stringent wash. Use protease to digest the
protein that is not covalently bond to the RNa molecule. Then reverse transcribe.
It gets to the amino acid and then stop and then sequence what comes out, the
more RNA that was bound to protein then the more sequencing was made
You can get a position of specific amino acid links
Pulling on the RNA and looking for protein and then Mass specto see what is
there. ICLIP – get resolution of nucleotide, sensitive
[RNA Immunoprecipitation] Pulling on PROTEINS, sequence the RNA.
Genome wide.You have bead and antibody and then you have an RNA binding
protein with the antibody. Wash of unbound stuff and then elute RBP-RNA
complex and then RT-PCR to ask what specific RNA was bound or deep
sequencing.Then traditionally, doesn’t involve crosslinking You get a lot of extra
stuff when you don’t crosslink. [RNA Seq] In vivo: transcription and then
splicing to mature RNA. In vitro: fragmentation, RT, and ds-cDNA fragments
followed by high-throughput-sequencing. In silico: sequence processing and
alignment to get genome sequence.To analyze continuously changing cellular
transcriptome and can look at alternative gene spliced transcripts, post
transcriptional modification, gene fusion, mutations, expression voer time. Exonintron boundaries. [Gel Shift] You radiolabel the RNA, control lane with no
protein to see where RNA was, first few are just how the RNA moves in the gel
for comparison. You add increasing concentrations of proteins.. With that you
should get increase binding and an increase shift. Evidence for interaction
between RNA and protein. [Competition] To see if interaction is specific. Take
the largest concentration complex. Then add in excess of cold RNA of the same
sequence in increasing amounts and then see an inverse of the gel. If you see
random RNA interrupts the interaction, then it is nonspecific. [Chemical
footprinting assay and primer extension]
Uses many compounds that attack RNA (create covalent bonds with RNA), then
you do a reverse transcription (“primer extension)
If the RNA molecule has been modified, then the reverse transcription will stop
(then you can see on a gel where RT stopped)
Bottom band of gel is the primer alone
Each band above is the an increased nucleotide
Always a sequencing gel that tells you the nucleotide sequence, so you can figure
out exactly where on the sequence the RT stopped
Then ask what happens when you add the RNA binding protein
Then in the absence of a stop, that’s where you see that there is an RNA binding
protein; wherever, the RT was not stopped, but has a break in the bands (but still
continues). Using purified proteins with “naked” RNA [Yeast 3] Hook bait fish,
look for RNA-protein interactions of unknowns of each. Hybrid protein 1 and 2,
hybrid RNA, need interaction of all 4three parts for transcription and some color
colony. [In situ hybridization] Sample cells or tissues treated to fix target
transcripts in place and increase access of probe, which is either complementary
DNA or RNA. Excess is washed away and non-identical interactions wash away.
Then probe that was fluorescently labeled is localized and quantified in the tissue
using microscopy. Used to reveal the location of specific nucleic acid sequences
on chromosomes or in tissues, a crucial step for understanding the organization,
regulation, and function of genes
[Reporter Gene Assay] Regulatory sequence to be studied like a genes
promoter. There is a reporter gene upstream but close like a luciferase. Then the
DNA is transcribed to mRNA and a reporter protein is created and the amount is
easily measured by GFP by fluorescence. Determine strength of
promoter/enhancer, transfection efficiency, protein trafficking, protein protein
interactions. For dual, firefly and Renilla luciferase are both reported using the
same transcript and then having a ratio of activity.
[BoxB/lambaN/ MS2 tethering] Tether proteins to mRNA by using the 22 AA
RNA-binding domain in lambaN to tag protein of interest and its specific 19 nt
binding site (boxB) is inserted into the target RNA recruiting the properties of
the fusion protein to the RNA. Used to create mRNA-specific factors involved in
processes such as mRNA translation and nonsense-mediated mRNA decay.
Minimizes interference and has a small size are the advantages.
RNA STRUCTURE] Uracil instead of thymine which just has hydrogen instead
of methyl group, U and A but can have G-U wobble base pairs. More
possibilities of 2ndary sturucture formation in single strand. Ribose replaces
deoxyribose and more stable, which favors A-helix formation, In B-helix, the
2’OH of ribose would clash with phosphate. Can form hairpin, stem-loop,
pseudoknot. Non-canonical base-pairing, GU base triple and A-mior motif. U
[Translation]. When stop codon comes up, the RF comes into the A site. This
brings in water molecule that fuels hydrolysis of the peptide chain from the P site
into the cell. fMet-tRNAfmet in bacteria. tRNAfmet: charged by Met-RS, then
formylated by transformylase enzyme to protect the charged tRNA. In
eukaryotes there is no base pairing between the two. After this, conformational
change in the small subunit and IF2 hydrolysis. Eukaryotic mRNAs use scanning
for start site selection. eIF4e (cap binding protein binds the 5’ cap and recruits
additional Ifs. eIF4G interacts with PABP with circularization improving
efficiency of initiation. This complex recruits the ribosomal subunit and complex
scans mRNA from 5’ to first AUG. Scan needs many Ifs. Sequence context
affects start site selection in euakryotes. Can start at 2nd AUG in mRNA if first
has poor sequence consensus. eIF1 monitors base-pairing.When start codon is
selected, energy from GTP hydrolysis is used to promote subunit joining and
removal of IFS. Complex,difficult. Elongation cycle. tRNA selection. Decoding
– ribosome must distinguish between cognate, near-cognate and non-cognate
tRNA:anticodon interactions. By difference in free energy of binding between
correct and incorrect substrates. Non-cognate discrimination is easy, but near
cognate have small energy difference. More difficult. G is similar to A with
similar number of hydrogen bonds. Fidelity of tRNA selection is amplified by a
two-step proofreading mechanism. tRNA selection is divided into 2 phases by an
irreversible step: GTP hydrolysis by EF-Tu. Gives 2 chances to reject tRNA
before peptide bond formation, effectively using the same difference in free E
twice and multiplying the discrimination of the 2 steps. Must be in equilibrium in
order to take advantage of energy difference. Depends on induced fit mechanism
as well. If cognate and anticodon come together in decoding center, GTP
hydrolysis by EF-Tu is faster and tRNA rate of accommodation into large
ribosomal subunit is also accelerated. Ribosome recognizes codon-anticodon
interaction by monitoring the geometry of the codon:anticodon minihelix. H
bonding by rRNA nucleotides to codon-anticodon helix occurs. Any nonWatson-Crick base pair makes minor groove width incorrect for interactions. 3 rd
position monitored less stringently causing wobble to happen. Peptide bond
formation. Have P site tRNA contains electorphile, carbonyl. Amide of incoming
amino acid is the attacker, nucleophile. Base pairing caused by positoning of the
substrate by the rRNA nucleotides for tRNA-assisted catalysis of peptide bond
formation. The GTPase EF-G catalyzes tRNA:mRNA translocation.
Translocation on the small subunit requires the energy of GTP hydrolysis and
substantion conformaitonal changes on the small ribosomal subunit. EF-G is a
protein mimic of EF-TuctRNA complex. Termination. Begins when a stop codon
is decoded by a protein release factor. Ribosome recycling frees ribosomes for
another round of translation. After peptide relase RRF binds the ribosomedeacylated tRNA complex in the empty A site and recruits EF-G. RRF and EF-G
promote GTP-depenent release of mRNA and deacylated tRNA and subunit
dissociation. IF3 binds the small subunit and prevents subunit reassociation in
the absence of mRNA. Accuracy in translation: Aminoacylation: Synthetases use
a “double sieve” model (size, then proofreading). Ribosome does not check
accuracy of aminoacylation. tRNA selection: kinetic proofreading: 2 chances to
reject a near-cognate or non-cognate tRNA. induced fit: forward rate constants
accelerated by bind of correct substrate. Translocation must also be accurate (but
we know less about how this works). Class 2 release factors increase the
accuracy. In bacteria, they don’t do that, they are GTPase, doesn’t seem to be
critical
[Regulation of gene expression] Regulation of transcription is mainly by
regulation of initiation. Coactivators and corepressors affect interaction between
transcription factors and RNA pol. Insulators prevent inappropriate action of
regulatory proteins on nearby genes that should not be regulated. Insulator can
block off promoter b from interaction with the enhancer. In both bacteria and
eukaryotes, multiple regulatory sequences at a given provide combinatorial
control and signal integration. Different binding sites for gene X work in
conjunction with others and can work with a different combination of other
factors as well. In eukaryotes, you use regulons: Not transcribed as a single unit,
but regulated by the same protein(s) and thus coordinately expressed. In RNA as
well. In eukaryotes, chromatin modification plays a major role in the regulation
of gene expression. Transcriptional control in bacteria: Regulation by glucose.
Low [glucose] -> high [camp]. Camp binding to CAP increases affinity for CAP
operator site. The trp repressor is weak, so there is still weak expression.
Attenuation provides further control. Blocks RNA pol binding. When tryptophan
drops, then trp codons are stalled and then higher stability hairpin is formed and
out competes the attenuator structure. The Gal promoter (eukaryotes). Free Gal4
recreuits the SAGA complex which recruits mediator, acetylates histones,
recruits switch snf proteins that move histones out of the way. Certain sequences
at 5’ UTR promote decapping and then rapid 5’-3’ decay that DOESN’T depend
on deandenylation. Endoculeotic degradation initiated by endonucleolytic
cleavage, does not generally require deadenylation or decapping like RNAi. AURich Elements (AREs) in 3’-UTRs are hubs for regulation of mRNA decay.
Rapidly degraded under normal conditions. Bound by Tis-11 that destabilizes
and HuR, which stabilizes. Regulated by phosphorylation. [Translation
Regulation] Most regulation of translation happens at the level of INITIATION.
Global repression of initiation under conditions of amino acid starvation. AA
starvation induces shutdown of translation via inhibition of initiation. Happens
by phosphorylation of eIF2. Under normal, eIF2 binds GDP 400x more tightly
than GTP. Only GTP-bound eIF2 can bind Met-tRNA. Limiting amounts of
eIF2B act as the GEF that exchanges GTP for GDP on eIF2. Phospho-eIF2 binds
nearly irreversibly to eIF2B, blocking its GEF activity. Gcn2 phosphorylates
eIF2 under conditions of amino acid starvation. Other kinases like PKR, PERK,
HRI. A few genes that will escape this repression. Upstream ORFs allow
translation of specific mRNA under conditions of global translational repression.
The fact that they are encoded there is what is important, they will never be
anything important. Ribosome scans and starts translation at first AUG then stop
codon and translation stops. Initiation a lot less efficient under these conditions.
More likely just to scan through all the AUGs with how much it is
phosphorylated. Gene specific regulation of translation in bacteria usually occurs
through the 5’UTR. Through riboswitches ir translation repressor protein behind
start codon. Also temperature can hinder access to ribosome bind site. Gene
specific regulation of initiation in eukaryotes: More prevalent in eukaryotes than
in bacteria. Phosphorylation of initiation factors. 3’ UTR dependent translational
regulation: eIF4E-Binding Proteins (4E-BPs). 4E-BPS repress translation by
competition with eIF4G for binding to eIF4E. Blocks translation only at this
specific mRNA. Must be expressed at low concentration for when it is recruited
by a protein that binds that this specific mRNA. 3’UTR dependent translational
regulation: eIF4E-Homologous Proteins (4E-HPs). Recruited to specific mRNAs
through interaction with proteins at 3’UTR. Repress translation by binding
directly to the 5’ cap through a binding motif. Block initiation at later step:
Prevent subunit joining hnRNP K binds to 3’UTR not at high levels in the cell.
microRNAs are small noncoding RNAs that prepress translation and promote
mRNA degradation. Rrovides binding specificity to base pairing inhibit
translation and inhibition. Are 1 of 3 broad classes of Argonaute associated small
RNAs in eukaryotes: miRNA, siRNA (cleaves target RNA), and piRNA (down
regulate transcription) [Small noncoding RNAs] RNA interference is an RNAdependent gene silencing mechanism. Lin 4 inhibits activity of lin 14 thus
promoting program 2. Both lin-4 and lin-14 mutants fail to repress expression of
lin-14 protein in the L2 larval stage. L1 is when lin-14 is active and in wild tipe
in L2 the lin-14 is repressed. Lin-4 is thought to do that and repress it. Lin-14 is
protein coding gene. Lin-4 could base pair to all these sites in the Lin-14 3’ UTR
and is a small RNA that regulates the expression of the lin-14 protein. miRNAs
are genes with most of them from transcripts that only code miRNAs but also
introns can do that all well. Produced by a conserved processing machinery.
Transcribed by PolII, Processed by Drosha endonuclease to produce pre-miRNA
that is exported from nucleus. Cleavage by Dicer endonuclease in the cytoplasm
produces the mature miRNA, which binds to Ago/miRISC:Actions: mRNA
target cleavage, translational repression, mRNA deadenylation. miRNAs repress
gene expression by promoting poly(a) tail shortening. In embryonic cells:
repression of translation dominates. Elsewhere: mRNA destabilization is more
important. RNAi likely evolved as a defense against dsRNA viruses. dsRNA
fought back with evolution of RNAi suppressor proteins. piRNAs: Important for
germline development in a wide variety of organisms. Many function to repress
transposon expression/mobility. Functions include post-transcriptional repression
(similar to RNAi) and transcriptional silencing (by recruiting chromatin
modifiers) Most concentrated in germ cells, and found in sperm. Could inhibit
axon regeneration in C. elegans. Made in different ways in different organisms,
different from miRNAs and siRNAs. If you express a gene in the macronucleus,
then you also express piRNAs. These piRNAs hang out in the cytoplasm and
they are around when new macronucleus is formed. [Small regulatory RNAs]
<300 nt. Present in all 3 kingdoms (but details different), variety of mechanisms
Regulation, metabolite binding. Hfq mediates sRNA:mRNA interactions in
bacteria Present in at least 50% bacteria species. Sm family of proteins, ancient
also helped spliced eukaryotes. Loss of function: reduced fitness, impaired stress
response, Reduced virulence. Promote base pairing of small RNAs with target
mRNAs, required for regulatory output. Two faces that each bind RNA. Hfq
doesn’t directly have regulatory interactions, more of a catalyst for binding
reaction. Hfq associated small RNAs form regulatory “hubs” that respond to
environmental conditions. Kinda like riboswitches, inhibit translation, activate
translation, or activate degradation. Bind to ribosome binding site to block, or
some secondary structure in mRNA and then sRNA can bind to prevent 2ndary
structure from forming. sRNA can recruit polyA polymerase which adds A
which does NOT stabilize in bacteria. The action of trans-acting bacterial small
RNAs can be regulated. Competition for bind to Hfq or to the small RNA itself
can regulate activity of a particular sRNA. Circular RNA can act as a sponge for
mRNAs to prevent regular interactions [CRISPR] Direct repeats varying in size
from 21 to 37 bp, interspaced by similarly sized nonrepetitive sequences.
Clustered regularly interspaced short palindrome repeats (CRISPR). Blasted
spacer regions showed bombardment of invasive nucleic acids
Immune system was found and CRISPR provides acquired resistance against
viruses in prokaryotes. Aquired bacteriophage spacers in phage-resistant
mutants. Picks up any part of the bacteriophage. Like a conveyor belt that keeps
picking up new parts. Like a memory and old ones get lost from the 3’ end. Each
region produes a crRNA (CRISPR RNA) with a stemloop with a repeat from the
genome and gets loaded on the cas protein and uses crRNA as a guide to RNA
with similar sequence to degrade it. CRISPR systems are widely distributed in
bacteria and archae. Cas9: dsDNA endonuclease. First has to bind to CRISPR
RNA, then has to bind to DNA. Has REC Lobe to recognize and bind guide
RNA and DNA target. Nuc Lobe: responsible for cleavage with RuvC and HNH
nuclease domains. In the Cas9 guide complex, RNA base pairing to DNA is Aform and is allowed by unwinding of dsDNA. Guide RNA: binds to Cas9,
provides sequence specificity. PAM sequence: a short DNA sequence that must
be present in the target DNA adjacent to the guide RNA recognition sequence in
order for Cas9 to cut. Double stranded – recognized by protein:DNA interactions
(a arginines make base-specific H bonding interactions with major
groove)Epigenic regulation: Translational activation or deactivation [lncRNA]
Choice: How is one X selected for inactivation? Imprinted XCI: paternal X
always inactivated. Random XCI: equal chance of inactivating paternal vs
maternal.X chromosome dosage compensation in mammals depends on the long
noncoding RNA Xist. Xist represses Tsix when Xist is expressed and vice versa
Noncoding RNA (mRNA-like: capped, spliced, polyadenylated): Retained in
nucleus
Expresseed only from the inactive X becomes expressed from one X on
differentiation of female ES. NECESSARY for XCI (deletion of Xist locus leads
to loss of silencing). Sufficient for XCI (high-level expression of Xist transgene
on an autosome can induce partial inactivation of that chromosome). Induces
spatial reorganization of the X chromosome: creates a repressed nuclear
compartment that excludes the transcriptional machinery. Silenced genes are
recruited to this compartment. Xist RNA coats the inactive X chromosome. Xist
RNA recruits enzymes that deposit repressive chromatin marks. First jumps to
regions in 2D (cis) space, then spread out to seeding sites along the entire length
of chromosome. Happens more efficiently on chromosome not autosome. Xist
recruits protein complexes, including a polycomb group complex that deposits
repressive histone methylation. Expression of Tsix RNA is anticorrelated with
Xist expression. Xist spreading depends on the 3D conformation of the X
chromosome
Promoted by “booster” elements. LINEs may represent booster sequences. X
chromosome is strikingly LINE-rich. Efficiency of spread into autosomal regions
correlates with their LINE content. X inactivation is independent of Xist in
differentiated cells Models for Xist function in silencing: cage around
chromosome,. Conformational change in the chromosome that inactivates it
when in act of transcribing XIST RNA. XIST RNA, alone or interacting with a
nuclear factor, binds to the chromosome, inducing a conformational change that
inactivates. Opens a site inactivation machinery to attach. Binds to site on X that
binds another machine to inactivate
[lincRNA] Sequences of lincRNA is generally not conserved. But the chromatin
marks associated with their expression are. HOX gene family. HOX TFs
responsible for controlling the body plan. Head to tail axis. Transcriptionally
silent HOX regions are associated with heterochromatinization. In a line of the
chromosome, on left is headmost, right is expressed in tail. Expression of the
HOTAIR lincRNA is tissue-specific. Loss of HOTAIR results in transcriptional
induction of the HOXD locus. Using shRNA to knockdown and microarray data.
When expressing HOTAIR from HOXC cluster, regulates transcription in the
HOXD cluster. Many lincRNAs examined to date have roles in transcription
regulation: Decoys. Scaffolds. Guides. Enhancer
[RNA and Disease] Cis-acting disease alleles: splicing. Muscular dystrophy,
spinal muscular atrophy, dementia, . Pretty much the pre-mRNAs are spliced
incorrectly and there is one less exon for example (SMA). Cis-acting disease
alleles: premature stop codons. Result of mutation that you make a truncated
protein, very toxic. mRNAs with this are generally degraded by nonsense mutant
decay. NMD modulates disease. This can be protective against disease though.
Because possibly not producing the toxic protein because of these premature stop
codons. Cis-acting disease alleles: trinucleotide repeat expansions. Fragile X
syndrome: Expansion in copy number of nucleotide regions. Second most
frequent genetic cause of intellectual disability. Fragile site on the X
chromosome. Not quite X-linked pattern. An intermediate amount of repeats
actually produces more of the mRNA and resulting protein. Expressing repeatexpended FMR1 mRNA in Drosophila indicates the mRNA itself may be
important for disease pathology. FMR1P is an RNA binding protein. Conclusion
FMR1P can be pulled down using oligo(dT) beads. Panel A, in presence or
absence of RNAse. Panel B, in presence or absence of NaOH. Panel C, in
presence or absence of EDTA. Ribosomes cant associate in presence of EDTA.
FMR1P associates with translating ribosomes. Two Graphs. FRMTP regulates
local translation at synapses to mediate synaptic plasticity. Much of the
molecular mechanism of learning and memory is due to translation locally at
dendrites with FMRP being a part of the mechanism. In response to glutamate
receptors and the dendrites. Phosphylated FMRP inhibits translation, but then
dephosphylated to produce translation. Changing concentration of AMPAR at
the surface. Weakens synapses. Spinal muscular atrophy: Due to changes in
splicing. Autosomal recessive disorder resulting from loss of function of SMN1
(Survival of Motor Neurons): degeneration of motor neurons in spinal cord,
progressive muscle weakeness and paralysis. Second most fatal disorder.
Skipped exon 7 during splicing causes SMN1 loss, only SMN 2 remains. Transacting mutations that impair RNA metabolism have surprisingly specific disease
phenotypes. Review: pre-mRNA splicing by the spliceosome. Branch point
2’OH attacks 5’ splice site. The 5; splice site is now activated to attack the 3;
site. The intron is released from the spliced mRNA as a lariat. The spliceosome
is composed of snRNPs (and additional proteins). 5 snRNP complexes: SR
proteins that select splice sites and recognize splice site choice, RNA helicases,
and many others. SMN is required for proper assembly of snRNPs. SMN is
required for this complex that gets loaded on every snRNP and you need these.
Can’t have splicing without this whole complex as well. SMN2 mutation in
patients with better-than-expected phenotype. Splice enhancers and silencers are
cis-acting sequences that regulate the probability of a splicing event. Cis
sequences on the RNA that binds proteins that determines if splicing will occur.
On exon or intron. Smn2 mutation created an enhancer for exon 7 [RNA-based
therapeutic approaches] Antisense oligonucleotides (AOs): SR protein to ESE,
with a (SR)n-peptide and AO restores WT. snRNAs as vehicles for antisense
RNA:Have a snRNP and Sm core to attach to antisense and restore function
partially: RNAi: siRNA to degrade mRNA
Most available drugs are small molecules that target protein. Huge libraries of
small molecules and then identify proteins through research that inhibit or cure
disease and assay with the protein that you can do at large scale. Take assay and
throw compounds at it and look for molecules that have activity in terms of
inhibiting protein. Some protein families are “privileged” drug targets. Some
protein families may be more “druggable” than others.