genetics and molecular biology

1. M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2.  Processing of mRNA
 hnRNP
 snRNP particles
 5’Capping
 3’Cleavage and polyadenylation
 Splicing
 Pre-mRNA methylation

3.  There is essentially no
processing of prokaryotic mRNA,
it can start to be translated
before it has finished being
transcribed.
 Prokaryotic mRNA is degraded
rapidly from the 5’ end

4.  In eukaryotes, mRNA is synthesized by
RNA Pol II as longer precursors (pre-
mRNA), the population of different RNA Pol
II transcripts are called heterogeneous
nuclear RNA (hnRNA).
 Among hnRNA, those processed to give
mature mRNAs are called pre-mRNAs

5. Pre-mRNA molecules are processed
to mature mRNAs by 5’-capping, 3’-
cleavage and polyadenylation,
splicing and methylation.

7.  The hnRNA synthesized by RNA Pol II is
mainly pre-mRNA and rapidly becomes
covered by proteins to form
heterogeneous nuclear ribonucleoprotein
(hnRNP)
 The hnRNP proteins are though to help
keep the hnRNA in a single-stranded form
and to assist in the various RNA processing
reactions

8. 1. snRNAs are rich in the base uracil, which
complex with specific proteins to form
snRNPs.
2. The most abundant snRNP are involved
in pre-mRNA splicing, U1,U2,U4,U5 and
U6.
3. A large number of snRNP define
methylation sites in pre-rRNA.

9.  snRNAs are synthesized in the nucleus by
RNA Pol II and have a normal 5’-cap.
 Exported to the cytoplasm where they
associate with the common core proteins
and with other specific proteins.
 Their 5’-cap gains two methyl groups and
then imported back into the nucleus where
they function in splicing.

10.  Very soon after RNA Pol II starts
making a transcript, and before the
RNA chain is more then 20 -30 nt
long, the 5’-end is chemically
modified.
 7-methylguanosine is covalently to
the 5´ end of pre-mRNA.
 Linked 5´  5´
 Occurs shortly after initiation

11. 7-methylguanosine (m7G)

12.  Protection from degradation
 Increased translational efficiency
 Transport to cytoplasm
 Splicing of first exon

13.  In most pre-mRNAs, the mature 3’-end of
the molecule is generated by cleavage
followed by the addition of a run, or tail, of
A residues which is called the poly(A) tail.

14.  RNA polymerase II does not usually
terminate at distinct site
 Pre-mRNA is cleaved ~20 nucleotides
downstream of polyadenylation signal
(AAUAAA)
 ~250 AMPs are then added to the 3´ end
 Almost all mRNAs have poly(A) tail

15.  Increased mRNA stability
 Increased translational efficiency
 Splicing of last intron

16.  the process of cutting the pre-mRNA to
remove the introns and joining together of
the exons is called splicing.
 it takes place in the nucleus before the
mature mRNA can be exported to the
cytoplasm.

17.  Introns: non-coding sequences
 Exons: coding sequences
 RNA splicing: removal of introns and
joining of exons
 Splicing mechanism must be precise to
maintain open reading frame
 Catalyzed by spliceosome (RNA + protein)

18. Biochemical steps of pre-
mRNA splicing
Step 1: a cut is made at the 5′splice site,
separating the left exon and the right intron-exon
molecule. The right intron-exon molecule forms a lariat,
in which the 5′terminus of the intron becomes linked by a
5′-2′ bond to a base within the intron. The target base is an
A in a sequence that is called the branch site
Step 2: cutting at the 3′ splice site releases the free
intron in lariat form, while the right exon is
ligated (spliced) to the left exon.

19. C U R A Y
Lariat

20. Nuclear splicing occurs by two
transesterification reactions in which a free
OH end attacks a phosphodiester bond.

21.  Catalyzes pre-mRNA splicing in nucleus
 Composed of five snRNPs (U1, U2, U4, U5 and
U6), other splicing factors, and the pre-mRNA
being assembled
 U1 binds to the 5’ splice site, then U2 to the
branchpoint, then the tri-snRNP complex of U4,
U5 and U6. As a result, the intron is looped out
and the 5’- and 3’ exon are brought into close
proximity.
 U2 and U6 snRNA are able to catalyze the
splicing reaction.

25.  The final modification or processing event
that many pre-mRNAs undergo is specific
methylation of certain bases.
 The methylations seem to be largely
conserved in the mature mRNA.

27.  Alternative processing
 Alternative poly(A) sites
 Alternative splicing
 RNA editing

28.  Alternative mRNA processing is the
conversion of pre-mRNA species into more
than one type of mature mRNA.
 Types of alternative RNA processing
include alternative (or differential)
splicing and alternative (or
differential) poly(A) processing.

29.  Some pre-mRNAs contain more than
one poly(A) site and these may be used
under different circumstances to
generate different mature mRNAs.
 In one cell the stronger poly(A) site is
used by default, but in other cell a
factor may prevent stronger site from
being used.

30.  The generation of different mature
mRNAs from a particular type of
gene transcript can occur by
varying the use of 5’- and 3’-
splice sites in four ways:
(i) By using different promoters
(ii) By using different poly(A) sites
(iii) By retaining certain introns
(iv) By retaining or removing certain
exons

31. Alternative
splicing

33. (A) A cassette exon can be either included
in the mRNA or excluded.

34. (B) Mutually exclusive exons occur when
two or more adjacent cassette exons are
spliced such that only one exon in the
group is included at a time.

35. (C, D) Alternative 5’ and 3’ splice sites
allow the lengthening or shortening of a
particular exon.

36. (E, F) Alternative promoters and
alternative poly(A) sites switch the 59- or
39-most exons of a transcript.

37. (G) A retained intron can be excised from
the pre-mRNA or can be retained in the
translated mRNA.

38. (H) A single pre-mRNA can exhibit
multiple sites of alternative splicing using
different patterns of inclusion.