RNA PROCESSING Types of RNA Transcription of pre-rRNA Modification of pre-rRNA Trimming and cleavage Splicing of pre-rRNA Capping of pre-mRNA

1. RNA
PROCESSING
PRESENTED BY:
PRASHANT VC
DEPT OF ZOOLOGY
GUK

2. Introduction
 RNA : ribonucleic acid (RNA) is a polymeric molecule essential in various
biological roles in coding,decoding,Aregulation and expression of genes.
 Types of RNA : ribosomal RNA (rRNA), transport RNA (tRNA) , messenger RNA
(mRNA).
 Each of these RNA are transcribed and futher processed to become fully
functional in order to bring out certain biological functions
 Among them only mRNA goes under translation and remaining tRNA and rRNA
along with ribosomal proteins and amino acids carry out the translation process.
 After transcription all RNAs are in pre-RNA form and by the processing they are
converted into mature RNAs.

3. rRNA processing
 Transcription of pre-rRNA:
 rRNA is transcribed from rDNA (ribosomal DNA) in nucleolus.
 There are 4 types of rRNA found in eukaryotic cells 1. 5S rRNA 2. 5.8S rRNA 3.
28S rRNA 4. 18S rRNA.
 Ribosomes are made up of 2 subunits : 60S (composed of 5S , 28S and 5.8S
rRNA) and 40S (18S rRNA and 50 different proteins).
 Modification of pre-rRNA:
 This can be classified as different process occurs in different part of nucleolus.
 The early modification takes place on the dense fibrillar centre of nucleolus which
includes pseudouridation of adenine.
 Late modification takes place in granular components of nucleolus and it involves
methylation of nucleotides.

4.  Trimming and clevage:
 A long stretch of RNA is trimmed into two rRNAs : 1. 5S rRNA 2. 47S rRNA
by endonuclease and exonucleases.
 47S rRNA is precursor of 28S , 5.8S and 18S rRNA.
 Synthesis of 5S rRNA is done by enzyme RNA polymerase III and other
rRNAs are synthesized by RNA polymerase I.

5.  Splicing of pre-rRNA:
 the pre-rRNA sequence do contain some introns between to
exons therefore needs splicing.
 The sequence contains a conserved nucleotide adenine (A) on
which the 2` hydroxyl oxygen carrying lone pair of electron
attacks the phosphate of 1st exon-intron junction acting as an
electrophile.
 And the hydroxyl group becomes free on the junction which
attacks the phosphate group of 2nd exon-intron junction removing
a lariant.
 The two exons are joined together.

7. tRNA processing
 Transcription of pre-tRNA:
 tRNA genes are transcribed and processed in the nucleus but for further
charging they are sent to cytoplasm.
 From tRNA gene primary transcript is produced and different transcripts
are joined to make one pre-tRNA.
 There are 21 different tRNAs for 21 different amino acids.
 Clevage and trimming:
 5` end (5` leader sequence) and 3` end seq of the pre-tRNA is cleaved by
endonuclease enzyme further trimmed by exonuclease enzyme.
 These enzymes are Rnase p and Rnase E/F fir 5` end and Rnase D for 3`
end.

8.  Addition of CCA:
 At the 3` end of pre-tRNA a sequence of cytosine-cytosine-adenine is
added by the enzyme tRNA nucleotidyl transferase. The addition is called
quality control.
 Amino acids are loaded at the site of addition.
 Amino acids are added on 3` terminal of CCA by aminoacyl tRNA
synthetases to form aminoacyl tRNA.
 Modification of nucleotides:
 On average atleast 12 nucleotides are modified in each pre-rRNA.
 Examples :
 Adenine is converted to pseudouridine.
 Uridine is converted to dihydrouridine.
 Adenine is converted to inosine.

9.  Splicing:
 The pre-tRNA molecule contains introns which is to be removed out.
 The removal of the intron is performed by endonucleases such as
Sen34,Sen54, Sen2 and Sen15.
 On removal of intron one 2`3`cyclic phosphate group and 5`OH group is
exposed and are further joined with the help of
Phosphodiesterase,ligaes,kinase and phosphatase.
 On removal of intron pre-tRNA is converted into tRNA.
 Charging of tRNA:
 The charging of tRNA is actually addition of amino acids in the 3’ end
off tRNA.
 This process is done in the cytoplasm by aminoacyl tRNA synthetases.

12. mRNA processing
 Transcription of pre-mRNA:
 Pre-mRNA is transcribed and processed in nucleus and after formation of
mature mRNA it is sent to cytoplasm.
 Processing of Pre-mRNA basically involves three major processes:
1. Capping : addition of 7M guanine nucleotide on 5` terminal.
2. Polyadenylation: addition of polyadenine tail on the 3` terminal.
3. Splicing : introns are removed and exons are joined.
 After capping polyadenylation and splicing of pre-mRNA it is called as matured
mRNA.
 Mature mRNA is sent to cytoplasm fir translation.

13. Capping of pre-mRNA
 Capping of pre-mRNA means addition of 7me guanine nucleotide on the 5`terminal of
pre-mRNA.
 Capping starts as the transcript of mRNA emerges after transcription.
 Uncapped pre-mRNA possesses triphophaste group in the terminal nucleotide which is
to be capped (5`pppNpNn3`).
 Triphosphatase enzyme removes one phosphate group leaving two on the 5` terminal
nucleotide. (5`ppNpNn 3`).
 By the action of guanylyl transferase enzyme and GTP , guanine with one phosphate
group is added on the 5` terminal. (5`GpppNpNn3`).
 The guanine added in the 5` terminal is methylated on the 7th nitrogen by mRNA
guanine-N7-methione transferase. (5`7meGpppNpNn3`).
 In some cases further nucleotides are also methylated called as 2nd cap and 3rd cap.

15.  The CEC capping enzyme complex binds to polymerase II enzyme
as soon as the transcript emerges by transcription to bring out
cappong process.
 Importance of capping :
 Regulation of nuclear export.
 Prevention from degradation by exonucleases.
 Promotion of translation.
 Promotion of 5` proximal intron excision.

16. Polyadenylation
 Polyadenylation is the process of addition of multiple adenine nucleotides on the 3`
terminal of pre-mRNA.
 This process starts as soon as the termination of transcription is done.
 Just before some nucleotides at 3`end there is a cleavage sequence present AAUAA
which is recognised by CPSF ( cleavage and polyadenylation signal factor) and
CSF (Cleavage stimulating factor).
 These proteins binds toh the cleavage site and attract nuclease enzyme to cleave the
stretch of nucleotide after the sequence.
 On the free 3` end of pre-mRNA polyA polymerase enzyme adds adenine
nucleotides.
 A protein called polyA-binding protein ensures addition of enough adenine
nucleotides.

17.  In some genes the ckeavage proteins add a poly A tail at one of the several
cleavage sites.
 Therefore, polyadenylation can produce more than one transcripts (alternate
polyadenylation)to form a singke gene similar to alternate splicing.
 Importance of polyadenylation:
1. Polyadenylation provides stability to mRNA
2. Prevent degradation by exonucleases
 Exonucleases degradation the terminal nucleotides,to compensate muktiple
adenines are added so that if exonuclease degrades the nucleotides coding
sequences are saved.

18. 5`cap
Polymerase II
AAUAA
DNA
Cleavage signal
recognised by CPSF and
CSF
Nuclease activity
Poly A polymerase and polyA
binding protein (PAB)
AAAAAAAAAAAAAAAAAAAA 3`
5`
5`
5`
3`
3`
PAB PAB PAB
Pre-mRNA
Pre-mRNA
Pre-mRNA
Pre-mRNA
CSF
CPSF

19. Splicing
 There are several sequences on a pre-mRNA. Most introns starts with a
sequence of GU (splice donor site) and ends with AG (splice acceptor site).
 The presence of only these two sequences cannot confirm the presence of
mRNA. A conserved Adenine is always present 20-50 basepairs upstream of
acceptor site.
 A is followed by a polypyrimidine tract. The consensus sequence of branch site
is CU(A/G)A(C/U) where A is conserved.
 5’splice site: the exon-intron boundary at the 5’ end of the intron
 3’ splice site: the exon-intron boundary at the 3’ end of the intron
 Branch point site: an A close to the 3’ end of the intron, which is followed by a
polypyrimidine tract (Py tract).

20. 20

21.  The splicing is mediated by a group of proteins called snRNPs (Small nuclear
ribonucleo proteins). U1 U2 U4 U5 U6.
 This process can follow two mechanisms
1. Self splicing/ cis-splicing:
 Splicing in single RNA.
 Lariat shape
 Common
2. Trans splicing:
 Two different RNAs
 Y shape
 Rare ( c. Elegans and higher eukaryotes)
Spliceosome mediated Splicing

22.  Spliceosome is a group of snRNPs (small
nuclear ribonucleo proteins).
 U1 binds on the 1st exon-intron junction.
 A protein called BBP (branch site binding
protein) binds to the branch site (A) and
U2AF ( U2 auxiliary factor) which helps the
U2 snRNP to bind with branch site binds
with the BBP.
 A complex of U4,U5,U6 binds the the stretch
of intron and fold them to form a lariat.
 After the cleave of splice site these proteins
terminate lariat from the mRNA.
 And exons are joined to form a matire
mRNA.

24. Cis splicing / Self splicing
 This process takes 2 steps :
 Step 1 : The OH group of Conserved A
carrying lone pair of electron and acts as
electrophile and attacks the phisphoryl group
of G in 5` end of intron. 5` exons is released.
5` end of intron makes a three way junction.
 Step 2 : The OH group of 5` exon attacks the
3` splice site of intron. 5`exon and 3`exon are
joined and a lariat intron is removed
 Tbis process happens when there is one RNA
to be spliced.

25. Trans splicing
 Trans splicing is done when there are 2 or more RNAs to be spliced
together.

26. References
 Snustad, D. Peter, and Michael J. Simmons. Principles of
genetics. John Wiley & Sons, 2015.
 Allison, Lizabeth A. Fundamental molecular biology. Blackwell
Pub., 2007.
 Albert, Bruce. "Molecular biology of the cell." (2008).
 https://courses.lumenlearning.com/boundless-
biology/chapter/rna-processing-in-eukaryotes
 https://reactome.org/content/detail/R-HSA-72312

27. Thank-you