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Whats does mRNA start as in the nucleus ?



heterogeneous nuclear RNA (hnRNA)

-in nucleus
-heterogenous in size and sequence
-produced by RNA polymerase II; a copy of the gene
-has intervening non-coding regions-introns- which are spliced out, forming the smaller cytoplasmic form (exons are parts retained)
-the half-life of new hnRNAs is only a few minutes as they get processed into mRNA (as it is made, hnRNA is covered by small RNAs and proteins)


Pulse chase experiment:
experiment: processing of nuclear RNAs
expose cells to [32P]-labelled nucleotides for 30 min

-they purified nuclear RNA and separated according to size
-measure radioactivity in each size group
-large RNA molecules take up the label (80s, 50,000 bases)
*chase with unable phosphate for 3 hours- labeled fraction is much smaller now


pulse chase experiment conclustion

the primary transcript is made processed and shortened during that time


RNA splicing

-hnRNA, primary transcript
*made by RNA polymerase II
*doesnt last long, half life of 15 min, processed as it is made
-exon-contains the final coding sequence
-intron-will be spliced out
refer to slide 7 for mature, cytoplasmic mRNA pic


mRNA Splicing page 9

-introns much be removed with precision: one base off and the protein is wrong
*there are highly conserved, recognizable sequence regions called the splice-sites
-consensus sequence:same for almost all genes
*splicing is done by a number of protein/RNA molecules


mRNA splicing proteins/RNA molecules

-small nuclear RNAs (snRNAs) bind to proteins (the U Proteins) to form snRNPs- "snurps"
-the RNAs recognized the splice sites by base pairing
-the complex of snRNA, hnRNA and proteins is called a spliceosome
-spliceosome is formed in nucleus, as transcription takes place


spliceosome is comprised of :

1. snRNPs which are snRNAs bound to specific proteins (the U proteins)
2. other proteins that hold the assembly together


U proteins

recognize each other and bind to their substrates by base-pairing using their snRNAs


spliceosome u proteins accomplish the following

ligation of the hnRNA


Intron splicing process

1.hnRNA is substrate
2.U1 snRNA binds to the 5' side of the splice site by base pairing snRNA to substrate
3.U2 snRNA binds to certain region of intron, the branch point
4. the U4/U6 and U5 bind displacing the u1(splicosome is assembling a loop) U4 is displaced, U6 switches to bind the U2
5. Now U6 acts as an enzyme (cuts 5' splice site, cuts 3' splice site, simultaneously joins the cut exon ends together , releases the intron)
6. during the cutting process U5 has been holding the two ends


ribozymes -catalytic RNA

-in some eukaryotes (protozoans) as well as some mitochondria and chloroplasts, the splicing reaction is accomplished by RNA only(no protein)
-RNA is acting as a catalyst
-this ribozyme is actually a part of the intron
-it is likely that the catalytic function of the snRNPs is actually in the RNA portion (snRNAs)


messenger RNA structure

1. 5' cap of methylguanosine
2. a 5' untranslated region (5'UTR)
3. a continuous sequence of nucleotides encoding a protein
4. an untranslated region 3' to the coding region (3'UTR)
5.a tail of about 250 "A" nucleotides =poly A-tail