RNA Splicing, Capping, Poly-A tail

pre-mRNA recognition sequences as laid out

....AG/GU Pu AGUA........uacuuAucc.....PyNPyAG/G... exon/........................intron.............................../exon

poly-A tail roles

1. stabilizing the molecule during transport out of nucleus 2. early stages of translation

5' splice site

A/C+GGU (AG/GU or CG/GU) A/C +G is in exon GU is in intron

3' splice site

AGG (AG/G) AG in intron G in exon

After transcription:

RNA splicing Capping 5' end Poly-A Tail added

snRNPs

RNA-protein complex that removes introns and splices exons together

spliceosome

a multicomponent complex made of snRNA and proteins (snRNPs) that is required for splicing -most common way to cut out introns 1. U1 binds to 5' splice site 2. U2 binds to branch site 3. U4/U6 and U5 trimer connects U1 to rest of snRNPs 4. Intron looped out and exons brought together 5. 5' splice site cut; U6 holds on to end of exon 6. 5' site connects to branch site forming lariat 7. 3' splice site cut; lariat released and exons connect

poly-A polymerase

adds the A's to the end creating the poly-A tail

mature transcript

after introns are removed and exons are connected through splicing

alternative splicing

allows an organism to carry fewer genes in its genome; why humans make 150,000 proteins but only have 20,000 genes

ribozymes

an RNA molecule that functions as an enzyme, catalyzing reactions during RNA splicing involved in group 1 and group II intron splicing

co-linearity of gene expression

demonstrates that the DNA sequence in the coding strand is the same as the mRNA sequence which is usually same as amino acid sequence HOWEVER, not always colinear due to intron splicing

triphosphotase

first enzyme in the capping process that removes 1 phosphate

cap-binding roles

it's recognized by cap-binding proteins that help in: 1. movement of some RNAs into the cytoplasm 2. identifies transcript as mRNA 3. early stages of translation 4. splicing of introns

methyltransferase

last enzyme in the capping process that adds a CH3 (methyl group) to the guanine

guanyltransferase

second enzyme in the capping process that adds a genetically modified product (GMP) to the 5' nucleotide guanosine must be upside down

branch site

the adenine in the middle of intron

exons

the coding sequences of DNA can be big or small (1 nucleotide)

endonuclease

the enzyme in creating the poly A tail that cleaves at 20 nucleotides down from the polyadenylation signal (AAUAAA)

introns

the intervening sequences between exons that do not code for anything can be big or small but have a minimum

lariat

the looplike structure created in the splicing of nuclear pre-mRNA in which the 5' end of an intron is attached to a branch point in pre-mRNA

AAUAAA

the polyadenylation signal for endonuclease in process of making poly-A tail

primary transcript

the pre-mRNA, both exons and introns are transcribed

RNA splicing

the process that removes the introns in the pre-mRNA to produce the mature mRNA 3 mechanisms: 1. group I intron splicing (introns cut self out) 2. group II intron splicing (introns cut self out) 3. spliceosome

poly A tail

the string of adenine nucleotides at 3' ends after stop codon the A's are enzymatically added AFTER gene is transcribed; it is NOT encoded in the gene sequence AAUAAAA is the signal endonuclease receives to begin cleavage

capping

this occurs as the pre-mRNA is being synthesized by RNA polymerase II, usually when about 20-25 bases long most mature mRNA's have a 7-methyl guanosine covalently attached to their 5' end 3 enzymes: 1. triphosphotase 2. guanyltransferase 3. methyltransferase