Posttranscription/translatome

Question 1

RNA-binding proteins (RBPs)?

Answer 1

Highly-conserved major class protein family that bind to single or double stranded RNAs

Question 2

What are RBPs involved in?

Answer 2

Posttranscriptional regulation (stability, splicing, nuclear export, localization and localization of target mRNAs)

Question 3

What do some RBPs bind to?

Answer 3

The 3’ UTRs of target mRNAs recruiting cofactors involved in posttranscriptional regulation

Question 4

What is RIP-chip?

Answer 4

Immunoprecipitation of RBP and identify bound mRNAs with ORF microarray

Question 5

Puf3 usually binds…

Answer 5

3’UTR sequence in mRNAs of mitochondria genes

Question 6

mRNA targets of budding yeast Pumilio proteins (Puf1-5) are usually enriched in…

Answer 6

Biological function and cellular localization

Question 7

What is immunoprecipitation?

Answer 7

The isolation of a protein species from cellular extracts by a specific-binding antibody and separation of the complex from the remaining proteins by precipitation

Question 8

Describe the steps for immunoprecipitation (6 steps)

Answer 8

1. Lyse cells
2. Remove cell debris and isolate WCE
3. Add protein A/G beads coated in antibody and incubate
4. Wash and elute
5. Pull down magnetic beads
6. Downstream applications

Question 9

How do antibodies bind to proteins?

Answer 9

Via epitope tags

Question 10

What is an epitope tag?

Answer 10

Small proteins attached to your protein of interest by cloning to facilitate pulldowns/IP and purification
- Tags (e.g. HA, FLAG, Myc, V5 and GFP) have commercially available antibodies that have been tested (high specificity and strong binding)

Question 11

What do epitope tags allow for?

Answer 11

Bypasses the need to make an antibody for your protein of interest by injecting the protein/antigen into a rabbit to harvest the antibodies (no guarantee that the antibodies will be good)
- One antibody can be used for many experiments

Question 12

Describe the steps of a RIP, starting from IP (4 steps)

Answer 12

1. Add proteinase k and incubate to remove protein from RNA
2. Purify RNA
3. Reverse transcribe RNA to cDNA
4. Either sequence RNAs (RIP-seq) or load RNAs onto chip (microarray)

Question 13

What is CLIP and what is it used for?

Answer 13

• Many RNA-protein interactions tend to be highly transitory (comes in quickly, does its job, then leaves) so its hard to determine change in expression on microarray
• CLIP is a special modified RIP that addresses this
• RBPs are crosslinked to their bound protein in vivo prior to lysis

Question 14

Describe the steps for Western blotting (6 steps)

Answer 14

1. Load and separate samples from WCE and/or IP onto SDS-PAGE
2. Electrophoretically transfer fractionated proteins onto PVDF membrane
3. Block the membrane with neutral protein (BSA or milk casein) -> used to saturate the samples and fill up any interactions that a non-specific antibody would have with the protein of interest
4. Incubate the membrane with a primary antibody specific to the target protein
5. Incubate the membrane with HRP-labeled secondary antibody specific to primary antibody
6. Incubate the blot with chemiluminescent HRP substrate and expose to film

Question 15

True or false: On a Western blot, secondary and primary antibodies have to be from separate organisms because antibodies don’t recognize antibodies from its own organism

Answer 15

True

Question 16

Describe how Pufs were found to degrade mRNA targets

Answer 16

• Transcriptome profiling (RNA-sequencing) of puf3 deletion strain was done.
• Found that Puf3 mRNA targets including COX17 were mostly upregulated in the puf3 deletion relative to wildtype.
• For mRNA degradation, cofactors include Ccr4-Not (poly-A tail deadenylation), and Dcp1-Dhh1 (decapping) promote exoribonuclease degradation. Puf3 recruited these to the 3’UTR.

Question 17

IP vs input vs mock IP on Western blot

Answer 17

IP: Band appears due to specific binding of antibody
Input: Band appears because this is a positive control (protein of interest is in the cell to begin with)
Mock IP: No band (negative control) because non-specific antibody is used so nothing is pulled down

Question 18

Describe genome-wide analysis of alternative splicing

Answer 18

• 95% of human multiexon genes are predicted to undergo alternative splicing (AS)
• Full-genome detection of alternative splicing is done by AS microarray profiling or RNA-Seq
  Steps:
  Say tissue X lack alternatively spliced exon, and tissue Y contains the exon
  1. Get mRNA from first exon, second exon and region in between exons from tissue X.
  2. Get mRNA from first exon, AS exon, second exon, region between first exon and AS exon, and region between second exon and AS exon from tissue Y
  3. Hybridize on microarray, Cy3 cDNA from tissue X and Cy5 cDNA from tissue Y
  4. Greater Cy3 means more exon skipping of AS exon, greater Cy5 means greater exon including of AS exon

Question 19

Where does AS occur the most among tissues?

Answer 19

The brain and the testis because the brain requires lots of microregulation and the testis requires lots of transcription rewiring
- so both spliced bands show up for many genes when ran on a gel

Question 20

Describe an example of important alternative splicing in the brain

Answer 20

In humans, neuronal-specific splicing factor nSR100 is downregulated in autism patients, and nSR100 mRNA targets have abnormal splicing in neural microexons
- Knockout of mouse nSR100 results in multiple autistic features

Question 21

Describe a 3’UTR GFP-reporter shut-off system (tethered function assay)

Answer 21

• nmt41 promoter fused to GFP protein so when nmt41 is transcribed, it is transcribed with GFP (fluorescent)
• In puf3 OE: puf3 will degrade the 3’UTR much faster, so GFP levels decrease faster
• In WT: normal degradation rate of nmt41
• In puf3 deletion: longer time for GFP degradation (degrades naturally anyways), GFP stays fluorescent for longer

Question 22

What is RT-qPCR used for in terms of microarray data?

Answer 22

RT-qPCR measures the abundance of mRNA to validate expression of microarray data

Question 23

Describe how RT-qPCR works to quantify amount of DNA

Answer 23

SYBR green: fluoresces when bound to double-stranded DNA; does not bind to single stranded DNA
- So fluorescence intensity is proportional to amount of PCR amplicon/mRNA abundance
- Use stats from fluorescence curve to figure out exactly how much RNA was initially present

Question 24

Define the translatome

Answer 24

Looks at which mRNA is being translated and at what rate it is being translated
- because even though an mRNA is in the cell, it may not be translated at a given time (translational control)

Question 25

Polysome?

Answer 25

All of the ribosomes on an mRNA strand together at a given point

Question 26

What does ribosome profiling detect? How does it do this in general?

Answer 26

Detects which mRNAs are associated with the ribosomes
- Next generation sequencing of ribosome protected mRNA fragments

Question 27

What are the steps to ribosome profiling? (4 steps)

Answer 27

1. Ribosome covers short segment of mRNA (28 bp) while translating (resistant to RNases)
2. Polysomes are separated into monosomes by treating samples with RNases, followed by separation on a sucrose gradient
3. Identify mRNA sequences protected by ribosomes (ribosome footprint) by removing ribosomes after separation to leave just the mRNA that they were translating and doing library generation/high-throughput sequencing
4. Counting the number of ribosome footprints along an mRNA can estimate the degree of translation

Question 28

What does ribosome profiling in yeast reveal?

Answer 28

Ribosome profiling reveals that translation varies at specific stages of meiosis
- Translation control correlates with gene function and stage such as DNA replication genes (premeiotic S-phase) or recombination genes (prophase I)
- Ex: SPS1 translation is inhibited in meiosis II even though mRNA is present in prophase I