Co- and posttranscriptional & translational regulation in eukaryotes

Question 1

How much of the molecular mass of a eukaryotic cell is comprised of RNA?

Answer 1

67% RNA! and the majority (2/3) of RNA is ribosomal RNA. Then protein makes up about 25% and the remaining 18% is DNA and other stuff.

Question 2

What is the difference between genes and transcription units?

Answer 2

Gene=function and transcription unit=structure.

Given the vast quantity of DNA that appears to have little protein-encoding power and the fact that so much of this DNA resides right in the middle of functional genes (as introns), some scientists prefer to think in terms of “transcription units” rather than “genes.” A transcription unit is a linear sequence of DNA that extends from a transcription start site to a transcription stop site.

Question 3

There are three kinds of RNA polymerase in eukaryotic cells, which and what are they responsible for transcribing?

Answer 3

RNA pol I: ribosomal RNA (rRNA)

RNA pol II: mainly mRNA but also snRNA, siRNA and miRNAs

RNA pol III: mainly tRNA, but also some other RNA (5S rRNA, snRNA U6, 7S rRNA)

So all RNA polymerases are in some way involved in the protein synthesis, focus of the cell! All RNAs are produced in the nucleus and have to go through different processes to reach their mature form.

Question 4

How does the size distribution of genes relate to organism complexity?

Answer 4

The more complex the organism, the more size distribution, more variable numbers of exons/more interrupted genes.

Question 5

What is the main factor determining the size of a gene in eukaryotic organisms?

Answer 5

The overall length of a gene is determined largely by its introns, as exons are usually short, typically
encoding fewer than 100 amino acids. Introns have very variable length.

Question 6

Explain in detail how capping of mRNA works.

Answer 6

5’-capping of mRNA happens co-transcriptionally, when the RNA pol II is paused.

The proteins handling the capping are associated with RNA pol II to be ready once the start of the transcript is out. During 5’-capping, a guanine is added to the triphosphate of the 5’ end of the mRNA molecule, which results in a 5’-5’ end structure, which is very unique (3’OH-7mG-5’-ppp5’-Np-3’OH). Then, the cap is methylated and the cap is done and very stable, which protects the mRNA from degradation (crucial!).

Question 7

What is the terms intron and exon short for?

Answer 7

Intron - intragenic region
Exon - expressed region.

Question 8

What provides the specificity during splicing?

Answer 8

The recognition elements of introns in pre-mRNA are conserved sequences specifying exactly where the intron starts: 5’ splice site (SS) = GU, and the 3’SS = AG. In addition to that, there is a conserved sequence at the branch site that is needed for intron excision.

Question 9

Explain in short how introns are removed from pre-mRNA.

Answer 9

First, the RNA is cleaved at the 5’SS and the intron folds and forms a lariat by the 5’ end of the intron binding to the branch site (UACUAAC). The intron is released as a lariat when the RNA is cleaved at the 3’SS and the 3’ end of the first exon is connected to the 5’ end of the second exon, and the intron is then debranched and can be degraded.

Question 10

During intron removal, “transesterification” happens, what does this mean?

Answer 10

Transesterification = phosphodiester bonds “moving places”, as many bonds that are broken are made, so no energy is lost during this process.

Question 11

What is the machinery handling splicing called and what does it consist of?

Answer 11

The splicing machinery is called the spliceosome. The spliceosome consist of five small nuclear RNAs (snRNAs) that are associated with proteins and together are called snRNPs (“snerps”). Together with some additional splicing factors and proteins, they form the spliceosome (which has over 150 components).

Question 12

Explain the spliceosome assembly pathway in detail.

Answer 12

E (early) complex: The U1 snRNP recognizes the 5’SS and base pair to it.
A: The U2 snRNP recognizes the BS and base pair to it in the presence of ATP and interact with U1 (and splicing factors) which bends the RNA.
B1: The tri-snRNP consisting of U4/6 and U5 joins.
B2: U1 and U4 release, which forms the catalytic center of the spliceosome in which U6 base pair with the 5’SS and U2.
C1: The first transesterification happens, where the hydroxyl group of the A in the BS attacks the phosphate in the 5’SS and this cleaves the bond and the lariat is formed.
C2: The second transesterification reaction happens, in which the 3’SS is cleaved and the exons are ligated together.

Animation here: https://dnalc.cshl.edu/resources/animations/rna-splicing.html

Question 13

How is the 3’-end of the pre-mRNA generated?

Answer 13

In the end coding region, there is a termination sequence, AAUAAA, which is transcribed by the RNA pol. This sequence is recognized by a complex containing an endonuclease and a poly(A) polymerase. The endonuclease cleave the pre-mRNA downstream of the termination sequence and the poly(A) polymerase adds a poly(A) tail (~200 A residues) to the pre-mRNA. The cleaved end connected to the RNA pol is targeted by exonucleases that degrade it, which causes the RNA pol to fall off which terminates transcription. The poly(A) tail will associate with poly(A) binding proteins which facilitate transport and indicate that the mRNA is mature.

The termination mechanism is very similar for RNA pol I and III, but have different terminators.

Question 14

Why do we call RNA pol II the ”The mRNA factory”?

Answer 14

Because it connects transcription and mRNA processing, which makes it all very efficient. When binding to the promoter, RNA pol is already associated with capping factors and splicing components.

Question 15

tRNAs is processed in four ways, which?

Answer 15

• During tRNA processing, the whole unpaired 5’-end is released
• Splicing occurs here too, but with a different mechanism than for mRNA, which exposes the active amino acid codon.
• a CCA sequence is added post transcriptionally, similar to the poly(A)-tail
• lots of modifications happen to individual bases, like pseudouridylation Ψ.

Question 16

How does the splicing reaction in tRNA work in short?

Answer 16

tRNA splicing occurs by successive cleavage and ligation reactions. An endonuclease cleaves the tRNA precursors at both ends of the intron, and the release of the intron generates two half-tRNAs with unusual ends that contain 5′-OH hydroxyl and 2′,3′-cyclic phosphate. These ends fold together and are ligated by a ligase.

Question 17

How is rRNA processed?

Answer 17

rRNA give rise to many different products, there is no splicing but things are processed out.

Production of rRNA requires cleavage events that involves small RNAs called snoRNAs (small nucleolar RNAs - nucleolus=like an oil drop in the nucleus). that acts as guides by base pairing to rRNA and modifying them, which is recognized by endonucleases.

Question 18

Alternative splicing is a rule, rather than an exception, in multicellular eukaryotes. Name five different modes of alternative splicing.

Answer 18

• Intron retention
• alternative 5′ splice-site selection
• alternative 3′ splice-site selection
• exon inclusion or skipping
• mutually exclusive selection of the alternative exons
• combinatorial exon selection
• alternative promoter splicing
• alternative polyadenylation splicing

Note: remember that splicing occurs co-transcriptionally!

Question 19

Give one example of alternative splicing and it’s outcomes.

Answer 19

Sex determination in drosophila is determined by alternative splicing, alternative stop codons give rise to different proteins, one that give rise to a functional protein in females and unfunctional in males. The functional protein affects alternative splicing that leads to female differentiation and the absence of it in males lead to male differentiation.

Question 20

What two factors can be affected by alternative splicing and how?

Answer 20

1) Quality: changing the quality can lead to different function. e.g. the female/male differentiation in drosophila.

2) Abundance: affect the stability of the mRNA. e.g. a premature stop codon being included can lead to nonsense-mediated decay.

Question 21

What four different types of elements regulate splicing?

Answer 21

Intronic or exonic splicing silencers and intronic or exonic splicing enhancers.

The effect of splicing enhancers and silencers is mediated by sequence-specific RNA binding proteins, many of which may be developmentally regulated and/or expressed in a tissue specific manner. The rate of transcription can directly affect the outcome of alternative splicing.

Question 22

How does the Nova and Fox families of RNA binding proteins regulate splicing?

Answer 22

Nova: if bound upstream or on exon –> silencing and if bound downstream –> enhancing.

Fox: if bound upstream –> silencing and if bound downstream –> enhancing.

Question 23

Explain how trans-splicing works in short.

Answer 23

Splicing generally occur in cis between exons carried on the same physical RNA molecule, but can happen in trans, even though rarely. Trans-splicing occurs between complementary sequences on introns from different mRNAs, through a similar mechanism as regular splicing, but with the 5’SS on a SL RNA (small leader RNA) and the 3’SS on another mRNA (usually from a polycistronic transcript). The GU seq of the leader reacts with the branch site on the mRNA intron 3’SS which ligates the exons together.

Question 24

What are the three main steps in any polymerizing reaction?

Answer 24

1. Initiation: start/not start.
2. Elongation: pace can be changed but hard.
3. Termination: important to know when and how to stop, as too long can lead to unfunctional transcript.

Question 25

Which step in protein synthesis is the most important regulatory step?

Answer 25

Initiation! The easiest step to regulate is starting or not, straightforward.

Question 26

Explain how translation in eukaryotes works.

Answer 26

Initiation: The small subunit of the ribosome binds to the methylated cap, migrates downstream until it reaches the start codon on the mRNA (AUG) and recruits the large subunit of the ribosome. tRNA with methionine (AUG) binds and the translation is initiated (also with the help from the poly(A) tail binding proteins which are positioned close to the initiation complex to stimulate initiation)

Elongation: the ribosome moves along the RNA, extending the protein AA by AA.

Termination: The stop codon is reached and this causes the ribosome to disassociate from mRNA.

Question 27

The poly(A) tail plays a very important role in unfertilized mammalian eggs, which are lay unfertilized for a long time before fertilization. What role?

Answer 27

Metabolism of the poly(A) tail gives the opportunity to express different proteins at certain stages without transcription. By metabolizing the poly(A) tail, the mRNAs are not recognized. So by adding/extending/removing the majority of the poly(A) tail, the cells can regulate protein expression without transcription!

Question 28

So, what are the main functions of the eukaryotic mRNA cap & poly(A) tail?

Answer 28

They are both ”structural tags for mRNA function & recognition”, involved in translational control, RNA degradation/biogenesis and miRNA can target the poly(A) tail for repression either by cleaving or controlling the length of it.