Lecture 11 - miRNA's, Proteomics Flashcards
What does miRs stand for?
MicroRNAs
Answer the following wrt microRNAs
a) how many nucleotides long are they?
b) are they single or double stranded
c) these RNA are _______ RNAs (coding/non-coding)
d) Which enzyme transcribes them?
a) 21-24
b) single
c) non-coding
d) DNA polymerase II
Nascent transcript called _______, which is processed into ______ and then processed further into the mature _______.
a) miR
b) pri-miR
c) pre-mir
b, c, a
MicroRNAs bind to target mRNA, forming dsRNA. What is the result of this?
degradation of the mRNA or inhibition of translation
MicroRNAs are expressed in a _______ specific manner
a) gene
b) cell
c) tissue
d) organism
c
What are the 3 cell types that are regulated by microRNAs?
- proliferation
- apoptosis
- development
As we know, microRNAs are non-coding, but what does this mean?
It means that they do not produce proteins themselves and instead assist elsewhere
Binds target mRNA, forming dsRNA, which results in degradation of the mRNA or inhibition of translation. Which one is more common in this case
Inhibition of translation
What is this image showing
a) pri-miRNA
b) pre-miRNA
c) miRNA
a
What is this image showing
a) pri-miRNA
b) pre-miRNA
c) miRNA
b
What is this image showing
a) pri-miRNA
b) pre-miRNA
c) miRNA
c
Describe the lengths (number of nt for each of the following RNAs)
a) pre-miRNA
b) miRNA
c) pri-miRNA
a) ~60nt
b) ~22nt
c) various lengths
a) What is the function of Dicer
b) What is the difference btw miRNA and miRNA*?
c) What is the purpose of the mature miRNA?
a) it removes the stem-loop, leaving two complementary sequences
b) miRNA is the mature one while miRNA* is the non-functional strand
c) it blocks protein synthesis or causes mRNA degradation
Fill in the blank (select the ones that apply)
a) post-transcriptional regulation
b) post-translational regulation
c) RISC
d) DICER
e) miRNA
f) mRNA
g) degradation
h) block translation
a) yellow
d) orange
e) green
g) blue
h) pink
Which part is the mRNA, and which is the miRNA
miRNA: red line
mRNA is the blue and black line with the poly-A-tail
based on this image the miRNA (red lines) are only blocking the end UTR of the mRNA so how is this blocking translation?
While the image is shown linearly, the actual structure will be bent and folded on itself, influencing how ribosomes bind to it. This is how the miRNA is able to influence and, in this case, block translation
upregulation of a miRNA typically leads to the _______ (up/down) regulation of a particular protein
down
How many miRNAs are there in the human genome?
over 2500
Where are the 5 places that miRNAs can live within the genome?
- part of multimeric complex (part of a cluster with other miRNAs all working together as a single unit)
- singly (work indep of other miRNAs)
- within coding sequence of a gene
- within introns of a gene
- between genes
miRNA coding sequence can occur as part of a multimeric complex or singly. What does this mean?
Multimeric complex: part of a cluster with other miRNAs all working together as a single unit
Singly: work indep of other miRNAs
Nothing is being transcribed wrt the MCM7 gene, so what could these conservation peaks be representing?
miRNAs
miRNA facts
a) Each miRNA can regulate _______ genes.
b) A gene can be regulated by several ________
c) miRNAs typically target the _____’UTR (3/5) of target genes
d) Which types of genes would be regulated by miRNAs
a) hundreds of
b) miRNAs
c) 3
d) genes involved with development
Match the following fxn to the genes; Neu1, AIDA, CBS, TBX2
a) metabolism
b) development
Which ones would you predict involve miRNA
a) Neu1 + CBS –> no miRNA
b) AIDA + TBX2 –> yes miRNA
T or F - genes involved with cellular development are regulated by miRNA
T
T or F - genes involved with cellular metabolism are regulated by miRNA
F - only cellular development, apoptosis or proliferation
T of F - the only factor that dictates occupancy between the mRNA and the miRNA is the seed region
F - Flanking region influences this as well
T of F - the only factor that dictates occupancy between the mRNA and the miRNA is the flanking region
F - Seed region influences this as well
Which region of the mRNA dictates whether the miRNA occupancy will be efficient or not
flanking region
T or F - there is a positive correlation btw gene expression and protein expression
F
T or F - there is a negative correlation btw gene expression and protein expression
F
T or F - it is difficult to correlate gene expression with protein expression
T
Is there a good correlation between mRNA expression and protein expression? Why?
No, due to the halflives of the proteins themselves which skews the difference in levels between the mRNA and the proteins
define proteome
The identity and quantity of every protein in a cell at a given time
What are the two characteristics of proteins that allow us to separate them
- Charge: use isoelectric points (pH)
- mass: molecular weight
Isoelectric point?
the pH in which that molecule has an electric charge of 0
Mass spectrometry is based on the principle that proteins have both a ______ and a _______
mass and charge
What is the Preferred tool for determining the identity of proteins?
mass spectrometry
What are the 2 key components of mass spectrometry?
1) Proteins are broken down into peptide fragments
2) Peptide fragments are analyzed based on their mass to charge ratio
a) What is the equation for the mass-to-charge ratio?
b) What technique is this used for?
a) molecular weight of protein divided by its charge
b) mass spectrometry
T or F - mass spectrometry is both qualtitiatve and quantitative
T
Everything has a mass, so this is easy. Oxygen has a mass of 16 Da, Carbon has a mass of 12 Da. Therefore, CO2 has a mass of ______ Da
12+(2x16) = 44
Mass Spectrometry: What about charge? Amino acids can have a charge, eg Asn. Is this what they mean?
No, Molecules are given a charge by passing the molecule through some ionization stream.
In mass spectrometry, what is the charge typically?
+1
answer the following based on the mass spectrometer m/z ratio profile
a) What do the 16, 28, and 44 represent?
b) which one(s) is/are the molecular ion(s)?
c) which one(s) is/are the fragmented ion(s)?
d) What does m/z mean?
e) What is the typical value for z?
a) molecular mass of each of the ion
b) 44
c) 16 + 28
d) mass/charge ratio
e) 1
Propane and CO2 have the same molecular weight. But why do they have different m/z ratios?
They have different properties. Propane’s most abundant ion is different than CO2s
which enzyme is usually used to break down the proteins into peptides?
the protease tyrpsin
What are the 5 benefits of using trypsin to break down proteins into different-sized peptides?
- stable
- Works over a range of pH values and temps
- specific + consistent cleavage
- cuts frequently (produces ideal peptide sizes)
- produces autolysis peaks
What does this mean: “Trypsin produces ‘autolysis’ peaks (which can be used in MS calibrations)”?
the autolysis peaks means that the protease cleaves itself over time, generating specific peptide fragments over time
What is the point of mass spectrometry?
to identify unknown proteins
Put the following steps regarding mass spec in order (unknown protein)
a) By comparing the mass and charge of the different fragments in the mixture, the composition of peptide can be determined.
b) The peptide fragment is then compared to a database of all known proteins
c) Peptides of an identical size are isolated and further partially degraded by
bombardment with an inert gas
d) This results in a mixture of shorter peptide fragments
e) Peptide has to be ionized so that it can respond to the electrical field
f) Repeat with the next set of identical peptides
g) Peptides are submitted to an electrical field and sorted based on mass (m) and
charge (z)
e -> g -> c -> d -> a -> b -> f
Match the following
a) y ion
b) b ion
1) N-terminal end of peptide
2) C-terminal end of peptide
a) 2
b) 1
T of F - peptides are fragmented sequenctially
F - randomly fragmented
T of F - peptides are fragmented randomly
T
T or F - some peptide fragmetnes are more common than others
T
a) How many peptides are present?
b) Which peptide fragment is the most frequent/abundant?
c) Which peptide fragment is the least abundant?
a) 1 which is fragmented
b) 716.2
c) 1481.1
when fragmenting peptidese
a) Which bond type is easiest/ most common to break
b) When that bond is broken, what does it produce?
a) C=O and NH bonds
b) y ion (C-term) + b-ion (N-term)
Based on this image, what is the molecular weight of glutamate (E)?
559.2 - 430.2 = 129
What is the molecular weight of Leucine (L)
716.4 - 603.3 = 113.1
The peptide is essentially recreated by correlating differences in ______ with AA ______.
peptide peaks, molecular weights
What are the 2 ways to ionize peptides
a) MALDI
b) ESI
a) matrix-assisted laser desorption/ionization (use laser to add charge)
b) electrospray ionization (use spray to add charge)
Match the following to the image
a) Separation MS
b) Ionization
c) Activation
d) Mass Analysis MS
a) blue
b) yellow
c) green
d) pink
What are th 4 steps to mass spec
1) to ionize the peptides so they can respond to the electrical current
2) to separate them based on their mass and charge
3) Peptides of identical m/z ratios are selected and sent to the Activation chamber to be broken down into b and y ions
4) The b and y ions are then sent to the second Mass analyzer to determine the m/z ratios for the mixture of ionized protein fragments
What is this image representing?
a) Ionization
b) Separation MS
c) Actviation
d) Mass Analysis MS
a
What is this image representing?
a) Ionization
b) Separation MS
c) Actviation
d) Mass Analysis MS
b
What is this image representing?
a) Ionization
b) Separation MS
c) Actviation
d) Mass Analysis MS
c
What is this image representing?
a) Ionization
b) Separation MS
c) Actviation
d) Mass Analysis MS
d
define Size Exclusion HPLC
High-performance liquid chormatography: tech that separtes mol based on their size passing them through a column filled with porous beads (larger mol elute faster - don’t get stuck in pores)
what is the first thing you must do before you send off your protein mass to get analyzed via mass spec?
Separate the proteins (by mass or charge), or you’ll get too much information + noise back
Put the following steps of mass spec in order
a) ionization of peptides
b) digestion of peptides
c) Purify POI
d) separation of peptides in the mass analyzer
e) tandem mass spec to sequence peptides
c -> b -> a -> d -> e
what are the 3 ways to do a crude cellular extract of your POI
1) SDS/PAGE gel-cut out bands
2) 2D gel- cut out spots
3) liquid chromatography
What are the 5 types of mass analyzers used to separate peptides?
1) time of flight
2) quadrapole
3) magnetic sectors
4) Fourier transform
5) quadrapole ion traps
What are the 2 hybrid separators used in the tandem mass spec to sequence peptides?
1) quadrapole-quadrapole
2) quadrapole-TOF (time of flight)
Match the following to the image
a) ESI
b) MALDI
c) MALDI-TOF mass spectrometry
d) Quadropole filter
a
Match the following to the image
a) ESI
b) MALDI
c) MALDI-TOF mass spectrometry
d) Quadropole filter
b
Match the following to the image
a) ESI
b) MALDI
c) MALDI-TOF mass spectrometry
d) Quadropole filter
c
Match the following to the image
a) ESI
b) MALDI
c) MALDI-TOF mass spectrometry
d) Quadropole filter
c
Match the following to the image
a) ESI
b) MALDI
c) MALDI-TOF mass spectrometry
d) Quadropole filter
d
TOF?
time of flight
TOF (time of flight) separates the peptide fragments how?
Based on size, the smaller peptide fragments will ‘fly’ faster down the tube than the larger fragments
How do quadrupole filters work to separate peptide fragments
They contain four poles, which alternate currents. Smaller peptides will hit the poles faster than larger peptides (greater charge, which results in this being repelled more)
What does the Human PeptideAtlas database show?
information about the protein involved in ur gene of interest
What are canonical core proteins?
essential, highly conserved proteins that play fundamental roles in cellular function and structure
What AA does trypsin cut at?
lysines
PTM stands for
post-translational modification
__________ is only one of many types of PTM, but it is the most common. This occurs mainly on _______ types (number) of amino acids. It adds about ______ Da to the mass of the protein
phosphorylation, three, 80
What are the 3 AAs that PTM mainly occurs on?
1) Serine
2) Threonine
3) Tyrosine
a) How many PTMs are in all types of tissues?
b) What is the most common PTM?
a) 486324
b) phospho-Ser (177399)
Define
a) HTP
b) LTP
a) high throughput: large-scale, automated methods that generate vast amounts of genetic data quickly
b) Low throughput: smaller-scale, often manual/semi-automated methods that process fewer samples or genes simultaneously.
T or F - HTP is more accurate than LTP
F - LTP is slower but, in the end, more accurate due to it being manual
T or F - LTP is more accurate than HTP
T
Which ones show HTP (high throughput data), and which ones show LTP data?
Top: HTP + LTP
Bottom: LTP
What is the purpose of the database STRING?
it finds what other proteins interact with my POI
Genomics can be used for determining:
a) ______ + ______ where your GOI is expressed (eg., microarray,
RNA-seq)
b) changes in _______ of your GOI under different conditions (GEO Profiles)
c) _______TFs that might be involved in the regulation of your GOI (ChIP)
d) _______ modifications that might affect the expression of your GOI
e) if your GOI might be regulated by ________s (miR db’s)
f) if your protein has post ________ (translational/transcriptional) modifications
a) tissues, cells
b) expression
c) putative
d) epigenetic
e) translational
What are the 8 things genomics can be used to determine?
Genomics can be used for determining:
1) tissues, cells where your GOI is expressed (eg., microarray, RNA-seq)
2) changes in expression of your GOI under different conditions (GEO Profiles)
3) putative transcription factors that might be involved in the
regulation of your GOI (ChIP)
4) Epigenetic modifications that might affect the expression of
your gene
5) If your GOI might be regulated by miRNAs (miR db’s)
6) If the protein levels are consistent with mRNA levels
7) If your protein has post-translational modifications
8) the interactions of your protein with other proteins
