RR9: RNA Processing II

Question 1

where and by what are rRNAs synthesised?

Answer 1

synthesised by RNA pol1 in the nucleolus

Question 2

what is the nucleolus?

Answer 2

section of the nucleus devoted to the formation of rRNAs and other ribosomal components

Question 3

what is rRNA transcribed from?

Answer 3

repeated sections in the ribosomal DNA

Question 4

how is the pre-rRNA processed? and what is special about it

Answer 4

the same in all organisms
little bits are cleaved out which gives rise to an 18S RNA, a 5.8 and a 28S
the same sequences, no matter the organism, give rise to the same conserved rRNAs

Question 5

what does the rRNA processing require instead of snRNA?

Answer 5

snoRNA (small nucleolar RNA)

Question 6

what happens if you introduce ribosomal DNA in an organism?

Answer 6

done with drosophilia
introduce trans gene that will integrate in the genome with these particular repeats, and that is sufficient to give rise to a nucleolus
just having RNA pol I start generating RNA associated with these repeats will give rise to a large liquid liquid condensate that will eventually become like the nucleolus
The RNA that is made brings in everything that will become a nucleolus (sticky)
The nucleolus is a membrane less organelle

Question 7

how are pre-tRNAs modified to become mature tRNAs?

Answer 7

1. 5’ end of the sequence has to be removed, cleaved right away
2. In certain circumstances, there are regions that are spliced out, but not classic splicing (not all but quite a few tRNAs)
3. A CCA trinucleotide is added on to the 3’ end, plays a role in the linkage with the amino acil tRNA synthetases, assuring that it can participate in protein synthesis
4. a number of the bases ill be modified and substituted for a different type of base, and even some of the riboses are modified

Question 8

what are some RNA binding domains?

Answer 8

give proteins the ability to interact with RNA sequences
RRM (RNA recognition motif), PTB (poly pyrimidine tract binding protein), KH, RGG, pumilio/PUF domains

Question 9

what is the structure of RRM and how does it interact with the RNA?

Answer 9

made up of a few beta sheets that are enriched in positively charged residues and they interact with negatively charged regions of the RNA

Question 10

what does PTB interact with?

Answer 10

interacts with that conserved poly pyrimidine rich region in the introns

Question 11

how do cells identify where the splicing apparatus has to sit down and carry out its reactions?

Answer 11

they utilise RNA binding proteins

Question 12

what is the mechanism of the use of RNA binding proteins to summon the spliceosome?

Answer 12

• One of the critical proteins for that is U2AF, it will interact with the 3’ end of the intron that is supposed to be explicit
• 2 subunits: smaller subunit (35 kilodaltons) will interact with the nucleotides around that 3’ boundary of the intron, including the AG nucleotide
• The bigger subunit, 65 kilodaltons will interact with sequences around that poly pyrimidine region
• You define at least the 3’ end of the intron to be spliced out
• The specificity of U2AF relies on defining where the exons are, and those are defined by a family of proteins called SR proteins that interact with exonic splicing enhancers within the exon sequence themselves
• SR proteins are rich in serine and arginine
• they decorate exons by interacting with these sequences in the RNA
• By covering the exons in the sequence of a pre-RNA, it helps U2AF identify where that critical AG will be and then also the poly pyrimidine track, telling it where to sit down and distinguish the 3’ end of the intron
• the U1 snNRP will use the info provided by SR proteins decorating the exons in order to define where the boundary will be at the 5’ end of the intron to be excised
• The U1 snNRP will sit down over the sequence, including the GU at the 5’ end of the intro to be removed, because it will be near those SR proteins near the exon

Question 13

what is this whole complex called?

Answer 13

the cross exon recognition complex

Question 14

what does alternative splicing give rise to?

Answer 14

different RNA isoforms made at different times or in different tissues
Gives several proteins with different properties that all come from the same transcription units
One promoter: different mRNAs: different proteins

Question 15

how is alternative splicing regulated?

Answer 15

in a temporal and tissue specific manner

Question 16

what is an example of a gene that shows alternative splicing?

Answer 16

• Fibronectin gene is expressed in fibroblasts and hepatocytes
• The mRNAs and proteins are a little different and that reflects the function in each of the cases
• In the fibroblast, the mRNA has a number of exons that include EIIIB and EIIA, which specify domains that are very sticky
• Strong affinity for the plasma membrane, and it interacts with the extra cellular matrix and has an adhesive function
• The hepatocyte (liver cell) has fibronectin that does not have the sticky domains
• This form has to circulate in blood and has an important role in clotting —> as it circulates through blood you dont want it to stick to stuff

Question 17

what can alternative splicing explain?

Answer 17

how humans have so few genes but so many proteins

Question 18

how is sex determined in drosphilia flies? (generally)

Answer 18

• They have sexually dimorphic characteristic typical to each sex (female/male)
• controlled by a cascade of RNA binding events that lead to specific alternative splicing outcomes
• All regulated by one singular RNA binding protein called sex lethal
• Has to be turned on in a manner associated with a chromosome counting mechanism
• you have to dosage compensate because females have 2 X and males have 1 X (females inactivate one or males upregulate)
• chromosome counting takes place early to carry out this compensation
• with that, the activation of expression of a gene called sex lethal
• Sex lethal is only expressed in females, not in males

Question 19

what is the full mechanism of sex determination in drosophilia flies?

Answer 19

• very early, maternally controlled establishment promoter for sex lethal, only present in female flies
• Downstream of the chromosome counting mechanism that occurs very early in embryogenesis in female flies (shut off in male flies)
• The transcription of the sex lethal mRNA and then the protein very early in development, there is sex lethal protein only in females and not in males
• When zygotic transcriptions begins, the promoter is active
• When it comes to piecing together the exons on that sex lethal gene (present in both males and females), the sex lethal protein through its RNA binding capacity will sit down at the 5’ end of an intron between exon 2 and exon 3 through a specific interaction and it will block U2AF from interacting with the RNA at that point so that the exon 3 is not recognized as something that you’re going to splice it to, rather U2AF in female flies will splice exons 2 and 4 together, leaving out exon 3
• in males you have 2,3,4 together
• Exon 2 and 4 together will give rise to more sex lethal protein, this time its zygotic
• In males, they have an mRNA that has an in-frame stop in the early part of exon 3, so when the ribosomes get on that mature mRNA, get to that stop and drop off (do not make sex lethal in males)
• The zygotic sex lethal protein again will interact with the RNA on the transformer gene (tra gene)
• Tra is expressed in both males and females, but because females have Sxl, sxl will interact with the 3’ end of the intron to be removed between exon 1 and exon 2 —> block U2AF
• In females, exon 1 and exon 3 are spliced together, exon 2 is excluded, in males you have 1,2,3
• in females those exons 1 and 3 will produce the tra protein
• In males there’s another in frame stop at the beginning of exon 2, never make the tra protein
• the tra protein made in females flies will interact with 2 other proteins, Rbp1 and tra2
• Rbp1 and tra2 are SR proteins
• Together as a ternary complex (tra, Rbp1 and tra2) , they decorate exon 4 in a gene called double sex
• Dsx is a transcription factor that will activate genes important to define sexual characteristics
• Two forms of dsx —> male specific features and one for female specific features
• Because of that SR protein RNA binding exon decorating complex, interact with exon 4 and when the double sex mRNA is being formed, exon 4 is recognized as an exon that has to be included in the final mRNA product (bc of those SR proteins)
• In female flies, you have exons 123456 which will give rise to female sexual characteristics
• In males, exon 4 is not decorated, so the mature mRNA has 12356, which gives rise to male sexual characteristics
• Female double sex gene will block genes that give rise to male specific characteristics
• Cascade event that gives rise to the two forms of flies

Question 20

where was RNA editing first identified?

Answer 20

in plastids like mitochondria and chloroplasts

Question 21

what are the enzymes involved in RNA editing and the particular changes they bring?

Answer 21

the enzymes are called deaminases
* Adenosine gets converted to inosine or
* Cytosine gets converted to uracil
* Sometimes it’s regulated

Question 22

what is an example of a gene that undergoes RNA editing?

Answer 22

• this kind of regulated editing occurs in apolipoprotein B
• one of the major proteins involved in low density lipoproteins, the things floating around carrying cholesterol etc
• Moving lipids in and around the body and into cells
• The protein itself is synthesized in the liver and also in the intestine
• In the liver, its very large (4536 amino acids), plays its role in transporting lipids
• In the intestines, there is an in frame stop halfway through the protein, caused by a regulated RNA editing event that converts a cytosine in the RNA to a uracil
• This gives rise to UAA residues, which is a stop codon
• So, apolipoprotein B in intestines is only about half the size of the protein made in the liver
• Don’t understand why that happens but it is regulated

Question 23

what is the poly A signal? and what does it do?

Answer 23

a signal that was common to almost all DNA regions that would give rise to an RNA signal responsible for recruiting in particular proteins that would give rise to this cleavage and then enhance the formation of a poly A tail
AAUAAA present in the 3’ end of pre-mRNAs
aso a more downstream signal (a bit less defined) that includes G nucleotides or a stretch of Us

Question 24

what is the protein complex recruited by the Poly A signal?

Answer 24

The protein complex includes CPSF (cleavage poly adenylation specificity factor) and a few auxiliary proteins such as CStF and CFI, this complex binds the pre-mRNA and catalyses a cleavage of that RNA molecule at a very specific site

Question 25

what happens after that cleavage of the pre-mRNA takes place and why?

Answer 25

The moment the cleavage takes place, an enzyme will immediately start to add adenosine nucleotides: Poly A polymerase (PAP)
* has to happen immediately because that 3’ end is very labile and vulnerable to enzyme that are degrading RNAs in the nucleus

Question 26

how many nucleotides are added by PAP and why does it need another protein, which adds how many nucleotides?

Answer 26

• At first 12 adenosines are added, PAP is not super efficient, the process is relatively slow
• BUT, poly A binding protein (nuclear version of PAP, so PABPn) enters the complex and enhances the ability of PAP to add on many more adenosine residues and does so very efficiently
• Acts like a co factor
• Can add on up to 200 adenosine residues which provide protection against exoribonucleases

Question 27

in what direction do exoribonucleases work?

Answer 27

3’ to 5’ direction

Question 28

what is particular about histone mRNAs?

Answer 28

they are not poly adenylated but have the stem loop in the 3’ untranslated region that provides the same kind of protection against degrading enzymes

Question 29

what is the distinction in the transcripts that go in the sense vs antisense direction when they start from the Cpg island promoters?

Answer 29

• divergent transcription takes place on the CpG promoters, which are the most common
• If we look at the transcripts generated in the sense direction (directed by TATA boxes) vs antisense direction, transcripts that accumulate are on the antisense strand are shorter, and not that many
• Transcripts done on the sense strand are more stable, longer
• There is an enrichment of polyAdenylation signals on the transcripts that head off in the antisense direction -> will be degraded
• Less poly A signals in the sense strand, but more U1 snRNP sites and there is very few snRNP sites in the antisense transcripts
• Combination of both is responsible for the differential degradation of the antisense (rapidly degraded because have things that they shouldn’t have and dont have things that they should have) and stabilisation of the sense strand