Splicing

Question 1

where does the processing of mRNA takes place?

Answer 1

All 3 processing steps – 5’-capping, 3’-polyadenylation and SPLICING
take place in the nucleus, and only when they are complete, mature
mRNA is secreted from the nucleus into the cytosol via a nuclear pore.

Question 2

what is splicing?

Answer 2

splicing to remove introns (segments of noncoding

RNA) and bring coding regions (exons) together

Question 3

what are introns?

Answer 3

Introns (in-terrupting region)
- segments of DNA or RNA that do not code for polypeptides
- sequences interrupt coding sequence of mRNA
- size ranges from 1 kb to several kb
- all higher and many lower eukaryotic mRNAs have introns
- must be removed by splicing to form a coding mRNA
molecule

Question 4

Exons (ex-pressed region)

Answer 4

• The actual coding sequences that are interrupted by introns

Question 5

True or false

Only introns are removed form pre-mRNA

Answer 5

False

Depending on the cell type, different coding exons can be removed

Question 6

Negative control - splicing

Answer 6

the repressor protein binds to the primary RNA transcript in tissue 2, thereby preventing the splicing machinery from removing introns.
NO splicing –>amino acid sequence that does not fold properly–> inactive protein

Question 7

Positive control - splicing

Answer 7

positive control in which the splicing machinery is unable to remove a particular intron sequence without assistant from activator proteins

Question 8

Why introns have conserved sequence

Answer 8

because there’s only one spliceosome

Question 9

Conserved sequence related to intron splicing

Answer 9

GU (5’)- AG (3’)
“branch point” A base 15-45 b upstream of 3’splice site
ploy-pyrimidine tract upstream of 3’ splice site

Question 10

Spliceosome

Answer 10

The existence of conserved sequence features at splice junctions
suggested that there must be a cellular machine that recognises these
sequences and carries out splicing.
Spliceosome Comprises > 100 protein factors
plus 5 snRNAs

Question 11

What are the component of spliceosome?

Answer 11

5 small nuclear ribonucleoproteins are components of the spliceosome (U1, U2, U4, U5, U6).
Each of the 5 small nuclear RNAs within this
spliceosome is found in complex with a protein.

Question 12

snRNP

Answer 12

The complex between small nuclear RNA and a protein
is called small nuclear ribonucleoprotein (snRNP,
pronounced SNURP).

Question 13

Sites of complementation by small nuclear RNAs

snRNAs

Answer 13

GU (5’)

Branch point

Question 14

U1

Answer 14

Small nuclear ribonucleoprotein U1 is a complex of snRNA with 4 different
proteins. Small nuclear RNA is drawn in black. It has a site that is complementary to
the 5’ end of the intron (shown in purple in the crystal structure).

Question 15

Events in splicing

Answer 15

First events in the assembly of the spliceosome:
U1 forms base pairs with GU at the 5’ splice junction
U2 recognizes the branch-point site A
the U4-U5-U6 complex joins the spliceosome
The spliceosome catalyzes the removal of the intron
through two consecutive splicing steps

Question 16

In the first splicing reaction

Answer 16

In the first splicing reaction, the 5’ end of the intron is
attached to the conserved A (the one that we call the
branch point). This is the first transesterification step. Here, a reaction between the C2’ hydroxyl group of ribose in nucleotide A and a target nucleotide in the upstream exon I leads to the breaking of a 5′–3′ phosphoester bond at the end of this exon. One of the cut ends is joined to the ribose C2’ in nucleotide A, closing the intron circle.
In the second splicing reaction, the inton loop is cleaved off and the two adjacent exons are joined. This is achieved in a second transesterification step.

Question 17

second splicing reaction

Answer 17

3’-OH of the 5’-exon becomes a nucleophile, and
in the second splicing reaction, the intron loop is
cleaved from the 3’ splice site and the two adjacent
exon sequences are joined

Question 18

intercistronic region

Answer 18

An intercistronic region separates the RNA coding region for protein A from the coding region for protein B. It is between the genes.
distance varies from -1 to +40 bases

Question 19

Introns

Answer 19

An intron would split the coding region for one protein (the gene) into two. It is inside the gene.

Question 20

Did introns evolved from intercistronic region ?

Answer 20

It is thought that introns evolved from intercistronic regions, because
the latter are found in both eukaryotes (albeit rarely) and prokaryotes
whilst the former in eukaryotes only.

Question 21

WHAT IS THE CATALYST IN

SPLICING?

Answer 21

In this process, the snRNA acts as a ribozyme, an RNA molecule that functions as an enzyme.
• In a few cases, intron RNA can catalyze its own excision without proteins or extra RNA molecules.
• Not all biological catalysts are proteins!

Question 22

Can intron RNA catalyze its own excision?

Answer 22

In a few cases, intron RNA can catalyze its own
excision without proteins or extra RNA molecules.
Example of group I self-splicing intron

Question 23

group 1 self-splicing introns

Answer 23

In group I self-splicing introns, the OH of a free
guanine nucleoside or a nucleotide cofactor (GMP,
GDP, GTP) attacks phosphate at the 5’ splice site.
3’-OH of the 5’-exon becomes a nucleophile and
the second transesterification results in the joining
of the two exons.

Question 24

RNA world hypothesis

Answer 24

RNA must have been the genetic material in the first cells, where it both encoded genetic information and
catalysed biological reactions.
- RNA viruses store their permanent information in RNA
- Short polymers of ribonucleotides can be synthesized nonenzymatically
in the laboratory.
- RNA-directed protein synthesis may have begun as weak binding
of specific amino acids to bases along RNA molecules, which
functioned as simple templates holding a few amino acids
together long enough for them to be linked.
–This is one function of rRNA today in ribosomes.