5' cap
TRANSCRIPT
Why the cap was evolved only in Eukaryotes?
Loss of Shine- Dulgarno base pairing in eukaryotes which is a means of
directing the ribosome to the mRNA
Eukaryote use ‘cap’ as an alternative signal for directing translational
machinery to the mRNA
Appearance of 5’exorionuclease only in eukaryotes
Existence of capping enzymes only in eukaryotes
Specificity of Guanylytransferase
Structure of 7-
methyl
guanosine
cap
5’ to 5’ triphosphate bridge( phosphoanhydride bonds)
Methyl group on N-7 position
Co-transcriptional capping
Carboxy-terminal
domain
~30 nucleotide
Addition of guanine in
opposite direction
Structure and mechanism of the RNA triphosphatase enzymes
Mechanically and structurally they are diverse
Two families
The divalent-cation-dependent RNA triphosphatase-
Found in protozoa, eukaryotic viruses and fungi
The metal-independent class of triphosphatase enzyme-
Found in metazoa and plants
Structure and mechanism of the RNA
guanylyltransferase enzymes
Lysine side chain
It have two structural domains
N-terminal nucleotidyl transferae
C-terminal OB fold domain
It resembles DNA and RNA ligases
Structure and mechanism of the mRNA cap
(guanine-N7) methyltransferase
It’s structure has revealed that, enzyme contains
separate ligand- binding pocket for AdoMet(methyl
donor) and cap guanine(methyl acceptor)
The exception
RNA dependent RNA polymerase of Vesicular Stomatitis
Virus(VSV) incorporates GDP into the cap structure
Functions of m7G cap
Essential for the growth of eukaryotic cells
Marks the translational start site
Protects mRNA from degradation by 5’exoribonucleases
Methyl group facilitate translation
Participate in mRNA splicing and enhances its accuracy
Binds to nuclear cap binding complex to facilitate mRNA transport to
cytoplasm
Specificity of Gunylytransferase ensures that the caps are added only to the
intact transcripts and not to endolyticaly cleaved once.
Up regulators:
1.Myc
2.E2F1-cell proliferation transcription factor
3.Importin alpha
Down regulators:
1.Cdc34-ubiquitin-conjugating enzyme
2.SAHH-S-adenosylhomocystine hydrolyase
mammalian cells contain a surveillance machinery anchored by the DXO and
Xrn2 factors that will rapidly degrade incorrectly capped pre-mRNAs
Possible Target proteins:
1. Viral TAT protein
2. Mammalian PMIT enzyme
mRNAs with short poly-A tails are captured poorly by oligo-dT based methods
Recombinant human cap-binding protein eIF4E was used to capture mRNAs
on the basis of the 5’ cap structure
With mRNA capture by the 5’ 7-methyl guanosine cap provides a more
complete view of the transcriptome and can be used to assay changes in
mRNA poly-A tail length on a genome-wide scale.
Based preparation and sequencing of concatamers of DNA tags deriving from
the initial 20 nucleotides from 5’ end mRNAs CAGE
Allows high-throughout gene expression analysis and the profiling of
transcriptional start points (TSP), including promoter usage Analysis
ReferencesAmiya k. Banerjee, 5'-Terminal Cap Structure in Eukaryotic Messenger Ribonucleic
Acids, MICROBIOLOGICAL REVIEWS, vol.2 June 1980
Stewart Shuman, What messenger RNA Capping tells us about the eukaryotic
evolution, Nature reviews, MolCellBio, Vol. 3 Aug. 2002
Meigang Gu and Christopher D Lima, Processing the message: structural insights into
capping and decapping mRNA, Current Opinion in Structural Biology, 15:99–106, 2005
Victoria H. COWLING, Regulation of mRNA cap methylation, Biochem. J. (2010) 425,
295–302
Tomoaki Ogino and Amiya K. Banerjee Unconventional Mechanism of mRNACapping
by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus Molecular Cell 25,
85–97, January 12, 2007
Jeffrey Wilusz, Putting an ‘End’ to HIV mRNAs: capping and polyadenylation as
potential therapeutic targets, Wilusz AIDS Research and Therapy 2013, 10:31
François Ferron, Etienne Decroly, Barbara Selisko, Bruno Canard, The viral RNA
capping machinery as a target for antiviral drugs, Antiviral Research 96 (2012) 21–31
Michael D. Blower et al, Combining Different mRNA Capture Methods to Analyze the
Transcriptome: Analysis of the Xenopus laevis Transcriptome, PLOS ONE ,October
2013, Volume 8, Issue 10
Toshiyuki Shiraki, Cap analysis gene expression for high-throughput analysis of
transcriptional starting point and identification of promoter usage, PNAS December 23,
2003 vol. 100 no. 26