Lecture 4 Single cell gene expression

Question 1

Characteristics of mRNA?

Answer 1

• It is the molecule that transfers the information from the DNA to the protein
• It participates in translating the genotype into a phenotype
• It is a key determinant of protein abundance (not the only determinant)
• Changes in mRNA expression allow the cell to adapt to changing environments by responding to stimuli
• Several mechanisms control mRNA levels, such as transcription, splicing mRNA decay

Question 2

What are techniques to study mRNA expression?

Answer 2

Mainly by Bulk Analysis
• -Low-to-mid-plex techniques (e.g. 1-10 mRNAs):
-Northern blot: RNA extr. -> electrophor. -> transfer RNA to membrane -> labeled probes -> visualization on x-ray film
- Reverse transcription PCR (RT-PCR) and qRT-PCR: RNA -> DNA -> fluorescence for amount
• Higher-plex techniques (>10 mRNAs):
- DNA microarray (disadv: you need to know the sequence already, can’t find anything new)
- RNA-Seq

Question 3

What do these techniques miss?

Answer 3

• These techniques miss important information about:
o Cell-to-cell heterogeneity: You measure the average of the whole cell instead of the specific parts
o Spatial information (sub-cellular or within a tissue/organ)

Question 4

Why is it important to study gene expression in single cells?

Answer 4

Assays analyzing the average signal from many cells may not yield the desired result because the cells of interest may be in the minority—their behavior masked by the majority—or because the dynamics of the populations of interest are offset in time.

e.g. o Cells that are in different stages of division but would look averagely the same
o Gene expression could be lower or higher in certain cells giving a wrong average

Question 5

When is it important to study gene expression in single cells?

Answer 5

E.g.

• In cancerous cells
• Resistant bacteria

Question 6

What are single-cell gene expression HIGH THROUGHPUT analysis methods for RNA/protein expression? Advantages/disadvantages?

Answer 6

Single-cell RNA sequencing/proteomics coupled to mass spectronomy

Adv/Disadv:

• High throughput
• Not yet sensetive enough for low-copy (tens) number mRNAs/proteins
• Spatial information lost

Question 7

What are single-cell gene expression LOW THROUGHPUT analysis methods for RNA/protein expression? Advantages/disadvantages?

Answer 7

1) Single-cell protein/RNA imaging
(E.g. RNA fluorescent in situ hybridization/RNA reporters for live imaging or Tagging with fluorescent proteins/YFP reporter/immunofluorescence)

Adv/Disadv:

• Sensitive to detect low abundance mRNA’s
• Spatial information is preserved
• Low-throughput

Question 8

Single-cell RNA imaging: why is it important? What can we learn?

Answer 8

Many prokaryotic and eukaryotic mRNAs are targeted to specific regions of the cell
• Cells are highly compartmentalized
• Compartmentalization results from protein localization at the right place
• Some of this spatial information comes from localized synthesis
• Spatiotemporal control of proteins production
• Rapid and response to local stimuli
• Facilitate protein assembly
• Controls cell differentiation and migration

Question 9

How was spatial information accessed in the 80’s?

Answer 9

• In the 80s: in situ hybridization with radioactive probes
  o Cellular fixation: to preserve cell, shape and mRNAs (formaldehyde)
  o In situ hybridization: with isotopically labelled probes
  o Detection: photographic emulsion
  Develop Fix
  And observe by light-microscopy
  or electron microscopy

Question 10

What is a major limitation of of using radioactive probes?

Answer 10

time and resolution

o To detect signal the photographic emulsion is exposed for weeks/months
o Radioactive decay causes spreading of the signal onto the photographic emulsion
o Thus, localization of the point source is not accurate

Question 11

What is a new approach to accessing spatial information?

Answer 11

A new approach: Using fluorescent probes

-> Fluorescence allows signal isolation from the focal plane
- The first fluorescence in situ hybridization (FISH)
o Spots of yellow fluorescence on each sister chromatid of chromosome 1 of the human lymphoma

Question 12

What was the first evidence of mRNA localization by in situ hybridization?

Answer 12

Autoradiograph of chicken fibroblast cells hybridized in situ with 3H-labelled probes to β-actin mRNAs encoding the cytoskeletal protein Actin

Question 13

How do we perform FISH today ?

Answer 13

hybridizing DNA oligo’s to fluorophores. Hybridize this with mRNA. You can see them as spots. You can count them and do a 3D imaging and then a Gaussian fit to count the RNA’s, look at distribution, etc.

Can see a single molecule of RNA in the cell

Question 14

What can a multi-colour FISH be used for?

Answer 14

Examples:

• mRNA compaction determined by multi-colour single-molecule mRNA FISH
• > then you can see if the mRNA is in the cytoplasm or in the nucleus (colours on each other/pairs)

Question 15

What can multiplex FISH be used for and what is it?

Answer 15

Different colours are given to mRNA’s. E.g. one RNA = blue-yellow-green, another is red-orange. Then you can distinguish single RNA’s. Creating a color code. Measure transcription of different genes at the same time. However, the genes need to be known.

Question 16

What controls mRNA localization?

Answer 16

ZIP-codes: cis acting motifs that direct mRNAs for transport to appropriate locations within a cell or organism
(‘The precise transport and translation pattern of localized mRNAs is dictated by cis-acting elements present on the mRNA molecule. Motor proteins bind to them.)

Question 17

Where are ZIP-codes found?

Answer 17

o Are found in UTR or in the coding sequence

Question 18

What do ZIP-codes physically do?

Answer 18

o Cis-acting motifs form 3D structures that are recognized by RNA binding proteins (trans-acting factors: contribute to formation of RNP defines specifity) and molecular motors
(Cis-regulatory elements (CREs) or Cis-regulatory modules (CRMs) are regions of non-coding DNA which regulate the transcription of neighboring genes)

Question 19

So, what did smFISH reveal?

Answer 19

• smFISH revealed that mRNA is localized in sub-cellular compartments in many organisms.

Question 20

What kind of field did multiplexed smFISH start?

Answer 20

• Multiplexed smFISH started the field of in-situ transcriptomics.

Question 21

What is a RNP-complex?

Answer 21

RNP is vessicle complex formed between RNA and RNA-binding proteins (RBPs)

Question 22

Why do we study gene expression in living cells?

Answer 22

• To understand how cells dynamically respond to environmental changes by controlling mRNA and protein synthesis, localization, translation and degradation.
• To detect transient and rapid events that we cannot see in fixed cells
• If we study gene expression regulation with single-cell resolution we can measure population heterogeneity and cell-to-cell variability
• We can also follow a single cell or a single molecule over time

Question 23

What are the technical requirements that allow us to study gene expression in living cells?

Answer 23

• Fluorescent labelling of mRNAs in vivo
• Optically compatible sample -> low auto-fluorescence is good
• Sensitive cameras (CCD/ sCMOS)
• Rapid sampling rate (33 frames/sec~video rate)
• Wide-field fluorescence microscopes with high Numerical Aperture (NA) objectives

Question 24

What kind of systems allow imaging single mRNA’s in living cells?

Answer 24

MS2 or PP7 Systems
allow imaging single mRNAs
in living cells (Give rise to highly quantitative imaging)

Question 25

What kind of techniques do these systems use?

Answer 25

1) using RNA loops that you can insert at the end of the RNA of interest: are bound by RNA binding proteins and fuse to fluorescent proteins.

Question 26

What are other techniques that can be used?

Answer 26

1) fluorogenic RNA’s: will be fluorescent only under certain binding conditions
2) Molecular beacons: no modification of RNA, don’t need sequence, no organism that you can manipulate etc. Oligonucleotide with fluorophores with quencher. Quencher reduces fluorescence. When binding to DNA, quencher is far away, fluorescence.
3) Cas9/Cas13 system: dCas13-GFP + guide RNA = Cas protein with GFP @ target RNA (doesn’t work very well) But, doesn’t require modification of DNA/RNA.

Question 27

Where did the idea of the MS2 system come from?

Answer 27

o Scientist took inspiration of bacteriophage MS2. The Bacteriophage MS2 is an
icosahedral single-stranded RNA virus.

Question 28

How can MS2 and PP7 coat proteins be used to visualize mRNA’s in living cells?

Answer 28

o MS2 and PP7 coat proteins to bind to different RNA stem loops
 Can be used to tag endogenous mRNAs (Endogenous substances and processes are those that originate from within a system such as an organism, tissue, or cell)
- MS2 system can be used to monitor all the stages of gene expression

(This RNA reporter system utilizes (= maakt gebruik van) repeats of an RNA loop derived from the bacteriophage MS2, that are inserted in the 3’UTR of an mRNA of interest. Binding of the MS2 binding sites by the MS2 coat protein fused to GFP allows us to detect single mRNA molecules in living cells. With this technology we measured different stages of gene expression, form transcription , to mRNA export and more recently to translation.)

Question 29

What stages can the MS2 system visualize?

Answer 29

All stages of gene expression: (e.g. transcription, export, localization, translation and decay).

Question 30

What is current research focused on?

Answer 30

On improving methods that allow the simultaneous detection of mRNA and proteins in single cells.