RNA
History
• mRNA and tRNA worked out in the 1960s
• Purification of RNA lead to 23S, 16S, and 4S
species and accounted for 85% of all RNA
• tRNA accounted for about 15% of all RNA
• Hybridization experiments did not match DNA
• mRNA accounts for only 1-4% of RNA
Types of RNA
•
•
•
•
•
•
•
mRNA
tRNA
rRNA
snRNA
snoRNA
siRNA
microRNA
Primary RNA
• mRNA (messenger RNA)
– Transcribed from DNA
– Is then translated into protein
• rRNA (ribosomal RNA)
– Necessary for helping ribosomes make protein
(catalysis)
• tRNA (transfer RNA)
– Mediates amino acid recognition with codons
Other RNA
• snRNA – small nuclear
• snoRNA – small nucleolar
• microRNA – micro
• siRNA – short interfering
These RNA species have a variety of functions
and not all of them have been worked out yet.
RNA and Rfam
• Pfam is a database we discussed to determine
groups of proteins
• Rfam is a similar database for families of RNA
• www.rfam.sanger.ac.uk
tRNA and folding
• tRNA have a unique fold
• There are multiple
websites to determine
RNA folding
• http://rna.tbi.univie.ac.at
/cgi-bin/RNAfold.cgi
Ribosomal RNA
• Has a structural and functional role in
ribosomes
• Prokaryotes: 70S which is 30S & 50S, which is
16S, 23S, 5S
• Eukaryotes: 80S which is 45S which is 18S,
28S, and 5.8S
snRNA
• Found in nucleus
• Involved with splicing and telomer
maintenance
• 5 classes U1, U2, U4, U5, U6
snoRNA
• Modify both rRNA and snRNA
• 2 Classes
– C/D Box – methylate rRNA
– H/ACA Box – convert uridine to peudouridine in
rRNA
miRNA
•
•
•
•
Recent discovery
Small approximately 22 nucleotides
Function to down regulate protein expression.
http://www.mirbase.org/search.shtml
RNAi (siRNA)
•
•
•
•
Double stranded RNA
Small, artificial
Degradation by RISC and Dicer
Protective mechanism for plants and animals
mRNA
• Coding RNA
• Central Dogma “one gene – one protein”
– No longer valid
– Alternative splicing, regulation, introns, etc.
• Altered expression by:
– Region, development, environment, disease, gene
activity
mRNA
• Key research areas
– mRNA expression database
• There is a DNA database, but is an mRNA database
equally good?
• Potential problems with and mRNA database
• How to measure mRNA expression
mRNA expression
• Steady-state mRNA (polyA isolation)
– Transcriptional control
– RNA processing control
– RNA export control
– RNA surveillance control
est database
• How to study mRNA
– Highly unstable, need to make cDNA
• Turn mRNA into DNA with reverse transcriptase
• Clone into a vector
• Sequnce (this is called an express sequence tag EST)
– ESTs can be isolated from specific cells at specific
times under specific conditions.
– This can aid in finding when types of proteins MAY
be expressed