RNA Processing or post

transcriptional modifications
Primary transcripts is linear, RNA copy of a
transcriptional unit.
Primary transcripts of both prokaryotic and
eukaryotic tRNA and rRNA are modified by
cleavage of the original transcripts by
ribonucleases.
Prokaryotic mRNA is generally identical to
itsprimary transcript, whereas eukaryotic
mRNA is extensivey modified.

A. rRNA
r- RNA of both prokaryotic and eukaryotic cells
aresynthesized from long precursor molecule
called preribosomal RNA.
From single precursor molecule 28S, 18S and 5.8S
rRNA are produced in eukaryotes and 23S, 16S
and 5S rRNA are produced in prokaryotes.
The nucleases that cleave and trim these
precursors of rRNA and tRNA are highly precise.


Primary Transcript
B. tRNA
tRNA precursors are converted into mature
tRNAs by a series of alterations:
cleavage of a 5 leader sequence
splicing to remove an intron
replacement of the 3 -terminal UU by CCA and
modification of several bases
Transfer RNA Precursor Processing
The conversion of a yeast tRNA precursor into a mature tRNA
requires the removal of a 14-nucleotide intron (yellow), the cleavage of a
5 leader (green), and the removal of UU and
the attachment of CCA at the 3 end (red). In addition, several bases are
modified.
C. Eukaryotic mRNA
most extensively modified transcription
product is the product of RNA polymerase II:
The immediate product of an RNA polymerase
is sometimes referred to as pre-mRNA.
Most pre-mRNA molecules are spliced to
remove the introns.
both the 5 and the 3 ends are modified
1. 5 Capping
5 triphosphate end of the nascent RNA chain is
immediately modified.
First, a phosphate is released by hydrolysis.
The diphosphate 5 end then attacks the a-
phosphorus atom of GTP to form a very unusual 5
-5 triphosphate linkage. This distinctive terminus
is called a cap.
The N-7 nitrogen of the terminal guanine is then
methylated by S-adenosylmethionine.
Transfer RNA and ribosomal RNA molecules, in
contrast with messenger RNAs and small RNAs
that participate in splicing, do not have caps.
Caps contribute to the stability of mRNAs by
protecting their 5 ends from phosphatases
and nucleases.
In addition, caps enhance the translation of
mRNA by eukaryotic proteinsynthesizing
systems.
Capping the 5 End.
2. Poly-A tail
Pre-mRNA is also modified at the 3 end.
Most eukaryotic mRNAs contain a polyadenylate,
poly(A), tail at that end, added after transcription has
ended.
DNA does not encode this poly(A) tail.
Eukaryotic primary transcripts are cleaved by a specific
endonuclease that recognizes the sequence AAUAAA.
After cleavage by the endonuclease, a poly(A)
polymerase adds about 250 adenylate residues to the 3
end of the transcript; ATP is the donor in this reaction.

The role of the poly(A) tail is still not firmly established
despite much effort. However, evidence that it
enhances translation efficiency and the stability of
mRNA is accumulating.
Blocking the synthesis of the poly(A) tail by exposure to
3 -deoxyadenosine (cordycepin) does not interfere with
the synthesis of the primary transcript.
Messenger RNA devoid of a poly(A) tail can be
transported out of the nucleus. However, an mRNA
molecule devoid of a poly(A) tail is usually a much less
effective template for protein synthesis than is one
with a poly(A) tail.
Polyadenylation of a Primary Transcript
A specific endonuclease cleaves the RNA downstream of
AAUAAA. Poly(A) polymerase then adds about 250 adenylate residues
3. Removal of introns
Most genes in higher eukaryotes are composed
of exons and introns.
The introns must be excised and the exons linked
to form the final mRNA in a process called
splicing.
This splicing must be exquisitely sensitive: a one-
nucleotide slippage in a splice point would shift
the reading frame on the 3 side of the splice to
give an entirely different amino acid sequence.
Thus, the correct splice site must be clearly
marked.
The base sequences of thousands of intron- exon
junctions within RNA transcripts are known.
In eukaryotes from yeast to mammals, these
sequences have a common structural motif: the base
sequence of an intron begins with GU and ends with
AG.
The consensus sequence at the 5 splice in vertebrates
is AGGUAAGU.
At the 3 end of an intron, the consensus sequence is a
stretch of 10 pyrimidines (U or C), followed by any base
and then by C, and ending with the invariant AG.
Introns also have an important internal site
located between 20 and 50 nucleotides upstream
of the 3 splice site; it is called the branch site
In yeast, the branch site sequence is nearly
always UACUAAC, whereas in mammals a variety
of sequences are found.
The splicing of nascent mRNA molecules is a
complicated process.
It requires the cooperation of several small RNAs
and proteins that form a large complex called a
spliceosome
Splicing begins with the cleavage of the
phosphodiester bond between the upstream exon
(exon 1) and the 5 end of the Intron. The attacking
group in this reaction is the 2 -hydroxyl group of an
adenylate residue in the branch site. A 2 ,5 -
phosphodiester bond is formed between this A residue
and the 5 terminal phosphate of the intron. Hence a
branch is generated at this site, and a lariat
intermediate is formed.
The 3 -OH terminus of exon 1 then attacks the
phosphodiester bond between the intron and exon 2.
Exons 1 and 2 become joined, and the intron is
released in lariat form.

Splice Sites
Consensus sequences for the 5 splice site and the 3 splice site are shown.
Py stands for
pyrimidine.
Splicing Mechanism Used for mRNA Precursors.
Small Nuclear RNAs in Spliceosomes
Catalyze the Splicing of mRNA
Precursors
The nucleus contains many types of small RNA
molecules with fewer than 300 nucleotides, referred to
as snRNAs (small nuclear RNAs). A few of them
designated U1, U2, U4, U5, and U6 are essential for
splicing mRNA precursors. The
These RNA molecules are associated with specific
proteins to form complexes termed snRNPs (small
nuclear ribonucleoprotein particles); investigators often
speak of them as "snurps."
Spliceosomes are large (60S), dynamic assemblies
composed of snRNPs, other proteins called splicing
factors, and the mRNA precursors being processed
Spliceosome Assembly
U1 (blue) binds the 5 splice site and U2 (red) to the branch point. A preformed
U4-U5-U6 complex then joins the assembly to form the complete spliceosome
Alternative splicing
It is a widespread mechanism for generating
protein diversity.
The differential inclusion of exons into a mature
RNA, alternative splicing may be regulated to
produce distinct forms of a protein for specific
tissues or developmental stages.
recent estimates suggest that the RNA products
of 30% of human genes are alternatively spliced.

Alternative Splicing Pattern
A pre-mRNA with multiple exons is sometimes spliced in different ways.
Here, with two alternative exons (exons 2A and 2B) present, the mRNA can be
produced with neither, either, or both
exons included. More complex alternative splicing patterns also are possible