Regulatory RNAs in eukaryotes and single cell methods

Question 1

What is the mechanism of action of small non-coding RNA (ncRNA)?

Answer 1

Small ncRNAs act by giving effector proteins specificity. They guide the effector proteins to their targets with high specificity due to antisense interactions (base pairing). The effectors can perform a variety of actions, like inhibit translation, induce degradation or induce modifications.

Question 2

Non-coding RNAs act at many different levels of gene expression. Give two examples!

Answer 2

• long ncRNA are involved on chromatin modification level in X chromosome inactivation.
• snRNA is involved in the spliceosome machinery on a co- and post transcriptional level.
• microRNAs can act as repressors on protein level by cleaving poly(A) tails leading to repressed protein levels.

Question 3

What are the main characteristics of micro RNA (miRNA) vs small interfering RNA (siRNA)?

Answer 3

• both are 20-25 nt long
• both have Dicer-dependent biogenesis
• miRNA precursor: imperfectly base-paired RNA (endogenous), siRNA precursor: perfectly base-paired (long) dsRNA (endogenous or exogenous)
• miRNA is present in all major branches of eukaryotic organisms and in animal viruses, siRNA is present in most eukaryotes (but not S. cerevisiae!)
• miRNA regulates all kinds of pathways (estimation ~60 % of all human genes) and siRNA regulates endogenous gene expression.

Question 4

What is the main function of siRNAs (small interfering RNAs)?

Answer 4

siRNAs are bound to effector proteins that cleave mRNA, so their main function is to regulate expression on a post transcriptional level in eukaryotes.

Question 5

Explain the siRNA pathway (RNAi).

Answer 5

long dsRNA (exogenous or endogenous) is cleaved into smaller dsRNA by Dicer, loaded onto argonaut and associate with RISC complex which removes one strand –> base pairing to target mRNA –> cleavage of target mRNA which degrade it –> reduction of gene expression.

Question 6

Explain the miRNA pathway.

Answer 6

endogenous ds pre-RNA is cleaved into miRNA by Dicer, loaded into argonaut (another AGO than in the siRNA pathway) which associate with RISC which removes one strand –> base pairing (not full complementarity) to target mRNA –> deadenylation of poly(A)-tail or blocking ribosome from binding –> reduction of gene expression.

Question 7

What is the source of the dsRNA that becomes miRNA and siRNA?

Answer 7

siRNA: either exogenous (virus or administered etc.) or endogenous transcribed in the nucleus: e.g. convergent transcription (overlapping), bidirectional transcription etc.

miRNA: primary miRNA from nucleus that is processed to pre-miRNA, that is then exported into the cytoplasm.

Question 8

How does the strand selection in the RISC/ago complex work?

Answer 8

From the double-stranded RNA loaded into the RISC/Ago complex, the strand with weakest base pairing at the 5’ end is selected as ”guide strand” and the other strand (”passenger strand”) is degraded. The passenger strand in siRNA is cleaved and degraded, while in miRNA it’s degraded by another mechanism.

Question 9

small RNAs can also regulate on a transcriptional level, how?

Answer 9

both miRNA and siRNAs bound to effector proteins can go into the nucleus and affect transcription by RNAi:
- RNA polymerase pausing
- Can affect splicing
- Induces histone modifications (H3K9me, H3K27me)
- Induces DNA methylation (directly in plants, probably indirectly in mammals)
- Can be induced/promoted by defective/slow RNA processing

Note: This occurs co-transcriptionally (RNA-RNA interactions)

Question 10

What are the advantages of using single cell methods?

Answer 10

When we perform bulk methods, we get a lot of data, and often we work with averages, but since cells in a sample is not necessarily homogenous it can be advantageous to use single cell methods to get a true representation.

It’s also good to give you information about tissue specific information and subcellular localization(like where in the cell something is expressed).

Question 11

There are two main approaches to single cell analysis, which?

Answer 11

1. In situ detection of DNA, RNA, protein, metabolites etc. Basically “on site” in the cell looking for stuff with different techniques.
2. Cell sorting/selection + analysis of DNA, RNA,
   protein etc. (qPCR, sequencing, proteomics etc). Basically sorting the cells and when you have the cells sorted, you can analyze them further.

Question 12

Give two examples of in situ detection methods used for looking at heterogeneity and quantification of a sample and explain how they work in short.

Answer 12

• Flow cytometry: You have a sample with fluorescent labels on different cells that you run through a fluid filled tube thick enough for one cell to pass at a time, then you have a laser that illuminates one cell passing through at a time and detect the fluorescence and scatter. This allows you to look at the heterogeneity of the sample, what different types of cells you have and gives you a quantification of the sample.
• Microscopy: labeling cells with for example fluorescent labels and looking at them in a microscope. This give you a good overview over how the cells look and also heterogeneity, but it’s harder to quantify.

Question 13

What in situ detection method could you use to study when and where a sRNA is present in an embryo?

Answer 13

One method you could use is FISH, Fluorescent in situ hybridization (FISH) in the cell/organism. You add small fluorescent probes that hybridize with known sequences (or random) and add it to cells and look at them in a microscope. This method have high specificity and the signal intensity provides metrics for quantification. This gives you spatial and temporal information (where and when?) and allows for single-molecule detection. This can be useful if you want to see where a sRNA is located in a cell and where for example.

Question 14

What method would you use to see where in a cell a specific protein is expressed?

Answer 14

Immunohistochemistry, In which you fix the cells, add antibodies are raised against the protein and then add antibodies raised against the primary antibodies, coupled to something that can be visualized, e.g. a fluorophore or enzyme that converts substrate to product. This allows you to visualize where in the cells the protein is expressed! The downside of this method is that you can’t do live imaging since te cells need to be fixed.

Question 15

What method would you use to see when/under what conditions a protein is expressed?

Answer 15

Using reporter genes encoding for fluorescent proteins fused to the gene of interest/promoter would provide live imaging of when the gene of interest is expressed. When the reporter is expressed, you know that your protein of interest is also expressed.

Question 16

Reporter genes can be used to study both transcription and translation of a gene of interest, where in relation to the GOI do you put them?

Answer 16

Using reporter genes to look at transcription, you fuse it after the promoter of the GOI. When the promoter is active, the protein is transcribed. This gives you no info about where in the cell the protein is expressed, but provides info on what tissue the promoter is active in!

Translational reporter genes are added right next to the GOI which creates a fusion protein. This give you info on subcellular localization but might not function as the native protein as you have added something to it. You could fuse an epitope flag (short peptide) to the GOI instead, which shouldn’t affect function as much.

Question 17

Give one example of a enzymatic reporter gene.

Answer 17

Convert substrate to colored product:
- lacZ (β-galactosidase)
- luciferase
- GUS (β-glucuronidase) – plants, fungi, bacteria

Question 18

Name two methods used for cell sorting/selection.

Answer 18

• FACS (Fluorescence-activated cell sorting) which is like regular flow cytometry but with sorting, e.g. by
  charging the cells to collect in different compartments, which is a method used to make your sample more homogenous.

-Microfluidics: oil used to cut off sample in drops, and then detect what is in each drop.

Question 19

When method(s) would you use to study all sRNA in a cell culture?

Answer 19

First, lyse the cells and enrich for RNA then use shot-gun sequencing to get dsRNA of everything in the cells, then I would run it with SDS-page to separate by size, extract the fragments smaller than 30bp and sequence them, eg by Illumina. There you go, now you have the sequences for all sRNA in the cell.

Question 20

What method would you use to investigate where a transcription factor binds?

Answer 20

CLIP (cross-linking immunoprecipitation) + seq. So first, we add the TF to DNA, then fragment the DNA but keeping the TF bound. Then we treat with antibodies that bind to the specific TF, and add secondary antibodies with magnetic beads so that we can wash away all the fragments not bound to the TFs. Then we can sequence the bound DNA and map it to the reference genome to find where the TF binds!