RNA Processing

Question 1

What are nascent RNA transcripts?

Answer 1

The initial molecule of RNA produced. An exact complementary copy of the template strand.

Question 2

Describe the RNA processing of prokaryotes

Answer 2

Generally, in prokaryotes, no RNA processing is necessary because the nascent RNA is usually the mRNA

-few exceptions: some archaea which have been found to have introns

Question 3

Why do eukaryotes need RNA processing?

Answer 3

In eukaryotes, the nascent RNA transcript has to be processed to yield pre-mRNA and then mRNA (transported into the cytoplasm for translation to occur)

Question 4

How can prokaryotes conduct transcription and translation?

Answer 4

• Prokaryotic mRNAs are synthesized on the bacterial nucleoid in direct contact with the cytosol and are immediately available for translation
• The Shine-Dalgarno sequence near the 5’ end of the maRNA binds to a site on the prokaryotic ribosomal RNA (rRNA), allowing attachment of the ribosome and initiation of translation, often even before transcription is completed

Question 5

Why do Eukaryotes require RNA processing?

Answer 5

• in eukaryotes, the mRNA is produced in the nucleus and must be exported into the cytosol for translation
• Precursor mRNA (Pre-mRNA) may include introns, which must be removed before translation can occur
• There is no ribosomal attachment sequence like the Shine-Dalgarno sequence in prokaryotes
• Eukaryotic mRNA requires extensive processing before it can be used as a protein template
  
  • This RNA processing takes place while mRNA is still in the nucleus

Question 6

What is an exon?

Answer 6

Any segment of DNA that is eventually expressed, usually translated into amino acid sequences

Question 7

What is an intron?

Answer 7

Noncoding regions (sequence of nucleic acids) also called intragenic sequences

• Introns are intragenic, that is found between the expressed sequences in a gene
• Not translated into protein

Question 8

Explain the features of eukaryotic mRNA

Answer 8

• Monicistronic
• One promoter, one gene
• mRNA contain introns which are spliced out
• Size of introns is roughly correlated with complexity of the organism
• mRNA requires significant processing
• Eukaryote mRNA contains a 7-mG cap on the 5’ end and polyadenylation of the 3’ end

Question 9

Explain the features of mRNA in prokaryotes

Answer 9

• Prokaryotic mRNA may be polycistronic(cistron is an old name for ‘gene’)
• Polycistronic mRNA carries the information of several genes, which are translated into several proteins
• These proteins usually have a related function and are grouped and regulated together in an Operon (clustered genes)
• One promoter, multiple genes
• Transcription and translation can occur concordantly
• Prokaryotic mRNA are not modified at the 5’ and 3’ end

Question 10

What is RNA processing?

Answer 10

RNA processing is the collective term used to describe the molecular events allowing the primary RNA transcripts to become the mature RNA(mRNA)

Question 11

What is the function of RNA pol II?

Answer 11

Acts as an RNA factory

• RNA polymerase II carries a set of pre-mRNA processing proteins on its tails
• The C-terminal tail of the RNA polymerase II acts as a tether for processing proteins to “hop” on to so they can process the pre-mRNA to mature mRNA
• Capping factors will modify the 5’ end of the transcript after ~30 nucleotides have been synthesized
• Splicing proteins and 3’ end processing proteins are also found on the C-terminal tail

Question 12

What modifications to pre-mRNA to make mature mRNA?

Answer 12

• Capping of pre-mRNA immediately after initiation of transcription at the 5’ end
• Termination of synthesis is combined with polyadenylation of the 3’ end
• Removal of introns from pre-mRNA
• Splicing of exons to make mRNA

Question 13

Explain the Capping of pre-mRNA

Answer 13

1. Phosphatase: removes one phosphate from the 5’ end of the RNA
2. Guanylyl transferase: adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’)
3. Guanine-7-methyl transferase: adds a methyl group to the 7 position of the terminal guanine
4. 2’-O-methyl transferase: adds a methyl group to the 2’-O position to the next to last base on the 5’ end

Question 14

What is the 5’-methyl cap?

Answer 14

The 5’-methyl cap is characteristic of eukaryotic mRNAs

• Capping s co-transcriptional
• Capped RNAs are produced on RNA polymerase II transcripts

Question 15

What are the roles of the 5’-methyl cap?

Answer 15

• helping the cell distinguish between different RNA in the cell
• regulation of mRNA processing
• transport from the nucleus
• prevention of degradation by nucleases
• Promotion of translation in the cytosol

Question 16

How is modification of the 3’ end of RNA done?

Answer 16

Accomplished by several enzymes associated with RNA polymerase II that bind to specific sequences on the RNA

1. Cleavage and polyadenylation specificity factor (CPSF) binds to the hexamer AAUAAA
2. Cleavage stimulating factor F (CstF) binds the GU-rich element beyond the cleavage site
3. Cleavage factors bind to the CA sequence at the cleavage site
4. RNA is cleaved and the GU rich region degraded in the nucleus
5. Poly-A-polymerase (PAP) adds approximately 200 A nucleotides to the 3’ end produced by the cleavage
6. Poly-A Binding proteins(PABP) binds to the poly-A tail and assist in directing translation by the ribosome

Question 17

What are the functions of Poly-A tail?

Answer 17

• enhances the stability of eukaryotic mRNA

- regulates transport of eukaryotic mRNA into the cytoplasmic compartment

Question 18

How is removal of introns from a pre-mRNA molecule?

Answer 18

1. At the 5’ end of an intron is the sequence GU and at the 3’ end is AG (plus some other sequence)
2. 18-38 nucleotides upstream of the 3’ end of the intron is an A located within the branch-point sequence
3. Intron removal begins with a cleavage at the first intron-exon junction
4. The G at the released 5’ end of the intron folds back and forms an unusual 2’ to 5’ bond with the A in the branch point sequence
5. This reaction produced a lariat structure
6. Cleavage at the 3’ intron-exon junction and ligation of these two exons completes the removal of the intron

Question 19

How are introns removed by the splicesome?

Answer 19

1. U1 and U2 snRNP bind to the 5’ s0lice junction and branch point sequence, respectively
2. Interactions with U4/U6 snRNP and U5 snRNP binds U1 and U2 together to form a loop
3. U4 dissociates forming the active splicesome
4. The splicesome complex cleaves the intron from exon 1 at the 5’ splice junction. The free end binds to the A in the branch-point sequence (lariat)
5. The intron is excised by cleavage at the 3’ splice junction and exons 1 & 2 are ligated. SnRNPs are released

Question 20

What is Alternative RNA splicing ?

Answer 20

RNA can be spliced in different ways to produce different species of mRNA in a process called alternative splicing

-Alternative RNA splicing can produce different forms of a protein from the same gene

Question 21

What are the four patterns of alternative splicing ?

Answer 21

1. Optional exon
2. Optional intron
3. Mutually exclusive exons
4. Internal splice site

Question 22

What is an optional exon?

Answer 22

A pattern of alternative splicing where an exon is included or excluded

Question 23

What is an optional intron?

Answer 23

A pattern of alternative splicing where an intron may be included in the transcript or spliced out

Question 24

What are mutually exclusive exons?

Answer 24

A pattern of alternative splicing where an exon may be sliced out in one transcript and a different exon may be spliced out in another transcript

Question 25

What is an internal splice site?

Answer 25

A pattern of alternative splicing - a weak splice site might exist within an intron to provide a secondary site of splicing

Question 26

Explain the control of alternative RNA splicing

Answer 26

1. Alternative splicing can occur due to intron sequence ambiguity. This may be due to ‘weak’ splice site sequences and therefore splicing choice occurs by chance 2. Alternative splicing can also be directed by negative and positive control via proteins that bind to splice sequences and either repress or activate splicing at that site

Question 27

Explain sex determination in the fruit fly-drosophila

Answer 27

The primary signal for determining whether a fly develops as a male or female is the X chromosome/ autosome ratio. - 1 (2 X-chromosomes/ 2 sets of autosomes)= female - 0.5 (1 X-chromosome/ 2 sets of autosomes)= male Three genes products are responsible for transmitting information about the sex ratio to cells The Sxl( sex-lethal) gene produces an initial alternate transcript that produces an active SXL protein. This protein affects splicing of Sxl RNA and the RNA of transformer (tra) and doublesex (dsx)

Question 28

In male drosophila:

Answer 28

1. Sex-lethal (Sxl) and transformer (tra) constitutively spliced to form nom-functional proteins 2. As a result of non-functional tra protein, splicing of dsx RNA produces a protein that represses female differentiation genes

Question 29

In female drosophila:

Answer 29

1. In females a transient Sxl transcript is produced that results in a functional SXL protein. This protein binds to normal Sxl transcripts AND produces an alternate splicing that produces functional protein 2. SXL protein blocks splicing of one exon in the tra transcript resulting in functional protein 3. TRA protein activates a splice site in dsx that results in a protein that represses male differentiation genes

Question 30

What is RNA Editing?

Answer 30

Post-transcriptional alteration of nucleotide sequence of protein-coding regions of transcripts: -A process in which information changes at the level of mRNA

Question 31

What is RNA editing revealed by?

Answer 31

- Is revealed by situations in which the coding sequence in an RNA differs from the sequence of DNA from which it was transcribed - In mammalian cells, there are cases in which a substitution occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded - In trypanosome mitochondria, more widespread changes occur in transcripts when bases are added or deleted

Question 32

Describe RNA editing in mammals

Answer 32

- In mammals, the apo-B gene encodes two alternative forms of the apolipoprotein B: Apo-B100 (liver) and Apo-B48 (intestine) - In the intestine, the apo-B mRNA is edited so that a premature stop codon is produced (CAA —> UAA) leading to the synthesis of the shorter Apo-B48 - this editing is accomplished by a cytidine deaminase enzyme

Question 33

What are the stop codons in translation?

Answer 33

UAG- you are gone UGA- you go away UAA- you are away

Question 34

Describe RNA editing in Trypanosome mitochondria

Answer 34

- In trypansomes, editing is more extensive and involves complimentary base pairing by a “guide RNA” - the editing is accomplished by an enzyme complex containing an endonuclease (cleavage), a terminal uridyltransferase (adding U), and an RNA ligase - Mitichondria has two mitochondrial genomes, one that has the sequence information to correct the other

Question 35

What are the steps of RNA editing in Trypanosome in mitichondria?

Answer 35

1. Guide RNA complimentary base pairs with target RNA 2. Endonuclease cleaves RNA at region of mispairing. 3. TUTase inserts uridine 4. RNA ligase ligates substrates RNA

Question 36

What is cryptic splice site?

Answer 36

Cryptic splicing signals are nucleotide sequences that closely resemble true splicing signals

Question 37

Explain the importance of a large mRNA through the nuclear pore complex

Answer 37

To produce an active protein: 1. The pre-mRNA must be processed into a messenger RNA 2. mRNA translocated from the nucleus to the cytoplasm 3. mRNA translated by the protein-synthesizing apparatus Newly synthesized RNA is packaged by proteins involved in post-transcriptional modification mRNA moves through the nuclear pore with the help of nuclear transport receptor proteins