RESNA N K

MICROBIOLOGY
RNA PROCESSING
 RNA molecules newly produced after
transcription – RNA transcript.

 RNA processing mean all the chemical

modification to generate a final RNA product.

 Bacterial mRNAs are exception.

 Primarytranscript of both tRNAs and rRNAs

must undergo series of processing in
prokaryotes as well as eukaryotic cells.
INTRONS
 Eukaryotic genes contain coding regions called exons.
 Noncoding regions called intervening sequence called
introns.
 Introns are common in eukaryotic genes but rare in
bacterial genes.
 Four types of introns present:

 Group I – found in some rRNA genes

 Group II – found in protein-encoding genes in
mitochondria and chloroplasts.
 Nuclear pre-mRNA introns – located in the
protein encoding genes in the nucleus
 tRNA introns – found in tRNA genes
Pre-mRNA Processing
 Eukaryotic mRNA is extensively altered after
transcription
 Changes are made to the 5’ end, the 3’ end, and the
protein coding section of the RNA molecule
 Modifications are done by
The addition of 5’ cap
The addition of poly(A) tail
RNA splicing
RNA editing
ADDITION OF 5’ CAP

 This capping consists of addition of an extra

nucleotide at the 5’ end and methylation of the
base of newly added nucleotide.

 5’ cap functions in the initiation of translation.

 It will increases the stability of mRNA.

 Methylation also takes place at 2’ position of the

sugar in the second and third nucleotides.
Addition of 5’ cap
Addition of 5’ cap
ADDITION OF POLY(A) TAIL
 Addition of adenine nucleotides at the 3’ end.
 3’ end is cleaved and polyadenylation takes place.
 Three important sequences beyond the end of the
coding sequence are involved in cutting and tail
addition.
 The tail signal (AAUAAA), a CA dinucleotide a few
base downstream, and a GU rich tract.
 In mammals polyadenylation requires complex
consist of poly(A) polymerase itself, CPSF , CstF , CF I,
CF II, and PABP.
ADITION OF POLY(A) TAIL
Addition of poly(A) tail in eukaryotic mRNA
mRNA with 5’ cap and poly(A) tail
RNA SPLICING
 Removal of introns.

 Occurs in the nucleus following transcription but before

RNA moves to the cytoplasm.

 Splicing requires three sequences:

 5’ splice site

 3’ splice site

 Branch point

 Most of the introns begin with GU and end with AG.

CHEMISTRY OF SPLICING
 Splicing is achieved by two successive
transesterification reactions, in which phosphodiester
bonds within the pre- mRNA are broken and new ones
are formed.
 The first reaction is by 2’OH of the conserved A at the
branch point.
 This group act as a nucleophile to attack the phosphoryl
group of the conserved G in the 5’ splice site.
 5’ exon is a leaving group in the transesterification
reaction.
 In the two reaction steps, there is no net gain in the
number of chemical bonds – two phosphodiester
bonds are broken , and two new ones are made.
Splicing reaction
Stucture of three way junction
THE SPLICEOSOME MACHINERY
 RNA splicing is carried out by a large complex called the
spliceosome. Complex comprises about 150 proteins and
5 RNAs.

 5 RNAs called small nuclear RNAs (snRNAs). They are

U1,U2,U4,U5, and U6.

 The protein complexes are called small nuclear

ribonuclear proteins (snRNPs)

 Some RNA-free proteins like U2AF (U2 auxillary factor)

and branch point binding protein (BBP) also involved in
splicing.
Some RNA-RNA hybrid formed during splicing
SPLICING PATHWAYS
 Spliceosome assembly begins with recognition of 5’
splice site by snRNP U1.

 Binding of splicing factor SF1(BBP) to the branch point to

form E’ complex.

 E’ complex converted to E complex by recruitment of U2

to polypyrimidine tract.

 SF1 replaced by U2 snRNP at branch point forming

splicoesome A complex.

 Further recruitment of complexes U4, U5, U6 tri snRNP

leads to formation of B complex .
 Extensive conformational change and remoddelling
incuding loss of U1 & U4 snRNP thus forming C
complex.

 Thus formed spliceosome complex then performs two

trans esterification reactions :

 In first reaction the 5’ end of the intron is cleaved

from the upstream exon and joined to branch A site .

 In second reaction the 3’ end of intron is cleaved

from downstream exon and the two exons are joined
together by phosphodiester bond

 The intron is then released in loop form and is

degraded.
Splicing pathways

B complex is converted
into C complex and then
removal of intron as a
lariat takes place.
 SF1

SF1

SF1
SPLICING PATHWAYS
SELF SPLICING
 Some introns are self splicing.
 They have the ability to remove themselves.
 Group I intron and Group II intron will spliced by
different pathway.

 In the case of group I introns RNA folds in a way that

forms a G-binding pocket which allows the molecules to
bind a free G nucleotide and use that to initiate splicing.

 Splicing of group II introns is accomplished by the

mechanism similar to spliceosomal mediated splicing of
nuclear genes.
ALTERNATIVE PROCESSING
 Here a single pre-mRNA is processed in different ways to
produce alternative type of mRNA.

 Results in the production of different proteins from the same

DNA sequence.

 One type is alternative splicing in which the same pre-mRNA

spliced in more than one way to yield multiple mRNAs

 Another type requires the use of multiple 3’cleavage sites.

 This will allow the pre-mRNA to be cleaved at different sites to

produce mRNAs of different lengths.
THERE ARE THREE MAIN AND ONE RARE TYPE OF ALTERNATIVE
SPLICING:

Alternative Promoter Selection

 occurs when two alternative promoters are utilized.

 The choice of which promoter to use depends on cell-

type specific transcription factors.
Alternative Promoter Selection
Alternative Tail Site Selection

 Is operative when alternative sites for adding the poly(A)

tail are possible.
 The choice between them again depends on cell type.

 In this case, cleavage at the earlier poly(A) site results in

loss of the distal exon.
 If the later poly(A) site is chosen, then the earlier poly(A)
site and the exon just in front of it are spliced out.
 This mechanism is used to produce antibodies which
recognize the same invading, foreign molecule but which
have different rear ends.
 One type of antibody is secreted into the blood, whereas
the other type remains attached to the cell surface.
Alternative Tail Site Selection
Alternative Splicing by Exon Cassette Selection

 Involves a genuine choice between actual

splicing sites.
 Depending on the choice made, a particular
exon may or may not appear in the final product.
 Here the primary transcripts are actually the
same.
 Some cell-type specific factor which recognizes
the different possible splice sites must come into
play here, but the details are still obscure.
Alternative Splicing by Exon Cassette Selection
TRANS-SPLICING
 Although rare, splices together segments from two
different primary transcripts.
 Trypanosomes are parasitic single-celled eukaryotes that
cause sleeping sickness and other tropical diseases.
 They evade immune surveillance by constantly changing
the proteins on their cell surfaces by the genetic trick of
shuffling gene parts.
 In addition they inhibit the trans-splicing of many
genes.
 On the other hand, trypanosomes do not appear to have
introns and so do not have normal splicing!
 Although it has not (yet!) been found in vertebrates,
trans-splicing of segments from one RNA molecule into
another also occurs in nematodes and in the chloroplasts
of plant cells.
Trans-Splicing
RNA EDITING

 All information about the aminoacid sequence

of a protein resides in the DNA is violated by a
process called RNA editing.
 Individual nucleotides in the interior of pre-
mRNA may be changed, added , or deleted by
RNA editing.
 In some cases, Guide RNA play a crucial role in
RNA editing.
 Amino acid sequence produced by the edited
mRNA is not the same as that encoded by DNA.
RNA EDITING
tRNA PROCESSING

 All tRNAs are similar in size and have a common

secondary structure known as cloverleaf.

 tRNA processing may include :

 Cleavage

 Splicing

 Base addition

 Base modification
 Transfer RNAs are transcribed as longer precursors that
also need processing.
 Some tRNAs are made singly, others are transcribed
together and in bacteria,some are included in the pre-
rRNA transcript.
 The 5’-end of bacterial tRNA is trimmed by
ribonuclease P.
 This enzyme is of note because it is a ribozyme.

 Ribonuclease P consists of both an RNA molecule and

a protein, but the catalytic activity is due to the RNA.
 The protein component merely modulates the activity
of the RNA.
tRNA processing
rRNA processing
In prokaryotes :
 The three rRNA molecules of bacteria are transcribed
together to give a single pre-rRNA.This contains 16S
rRNA, 23S rRNA and 5S rRNA joined by linker regions.

 In bacteria, this pre-rRNA transcript also includes some

tRNAs.
 The mature rRNAs are made by cleavage of the precursor
by ribonucleases. This occurs in two stages .

 First, internal cuts are made, separating the three rRNAs.

Ribonucleases III, P and F recognize sites where the pre-
rRNA is folded into double-stranded regions held
together by base pairing. Rnase D also involved.
 After this cleavage, the ends are trimmed by several
exonucleases.

 Methyl groups are added to specific bases and to the 2’

carbon atom of some ribose sugars.

In eukaryotes :
 There are four ribosomal RNAs. The 5S rRNA is made
separately and does not need processing.

 The other three (18S rRNA, 28S rRNA and 5.8S rRNA)
are made as a single pre-rRNA and processed much as
in bacteria.
rRNA processing in prokaryotes
Some pre-rRNA contain tRNA….
 rRNA processing in eukaryotes

Methyl groups are added to specific

bases and to the 2’-carbon atomof
some ribose sugars

The RNA is cleaved into several

intermediates

Mature RNA molecules

results
SPLICEDISEASE DATABASE:
LINKING RNA SPLICING AND DISEASE

 Juan Wang, Jie Zhang, Kaibo Li, Wei Zhao and

Qinghua Cui - Accepted November 12, 2011
 RNA splicing is an important aspect of gene
regulation in many organisms.
 Splicing of RNA is regulated by complicated
mechanisms involving numerous RNA-binding
proteins and the intricate network of interactions
among them.
 Mutations in cis-acting splicing elements or its
regulatory proteins have been shown to be
involved in human diseases.
 Defects in pre-mRNA splicing process have emerged as a
common disease-causing mechanism.
 Therefore, a database integrating RNA splicing and
disease associations would be helpful for understanding
not only the RNA splicing but also its contribution to
disease.
 The Splice Disease database provides information
including the change of the nucleotide in the sequence,
the location of the mutation on the gene, the reference
Pubmed ID and detailed description for the relationship
among gene mutations, splicing defects and diseases.
 They standardized the names of the diseases and genes
and provided links for these genes to NCBI and UCSC
genome browser for further annotation and genomic
sequences.
 For the location of the mutation, they give direct links of
the entry to the respective position/region in the genome
browser.
 The users can freely browse, search and download the
data in Splice Disease at
http://cmbi.bjmu.edu.cn/sdisease.
 Although the human gene mutation database (HGMD,
http://www.hgmd n.org/) integrated this kind of data but
there are big difference between HGMD and
SpliceDisease database.
 Firstly, HGMD is not free and only provides ‘search’
function for registered users for limited days.
 Secondly, HGMD only provides information of point
mutations of intronic sequence for splicing mutation.
 Thirdly, HGMD does not provide detailed descriptions
for the relationship among gene mutations, splicing
defects and diseases.
 On the other hand, Splice Disease database is a free
and comprehensive database containing cis-splicing
sequence mutations and trans-acting splicing
mutations that cause disease.
 At present, the ‘SpliceDisease’ database is at its first
step, it will be a valuable ongoing resource for the
study of RNA splicing and disease.
Splice disease result page: (A) once a user runs a search, there comes the result
summary page that includes nine item, (B) The direct link for entry to the
respective position of mutation. The sequence of exon shows in upper case and
intron shows in lower case. And one FASTA record per region (exon,intron)is used
in the sequence file. The intron/exon of the location of mutation is highlighted in
yellow color and specific nucleotide is marked in red color.
RESNA.N.K.
Ist SEM M Phil