Outline of Splicing
Mechanism of splicing
Em visualization of precursor-spliced mrna in an R loop
Kinetics of in vitro splicing
Analysis of the splice lariat
Lariat Branch Site
Splice site sequence requirements
Yeast Spliceosome
Human spliceosome
Alternative Splicing
Snurps or SnRNPs Small nuclear ribonuclear protein particles U1, U2, U4, U5 and U6 involved in nuclear RNA splicing Snurps constitute components of the spliceosome and are key components of splicing Each snurp has a unique role is splicing and the sequence of the RNA component of the snurp determines the role played in splicing
U1 interacts with the 5 splice junction: Mutations in the 5 Intron can be compensated by mutations in the U1 RNA
Mutations in the 5 intron can be compensated by mutations in the U1 RNA
The U6 snrnp also interacts with the 5 splice junction
U2 base pairs with the yeast branch-point sequences within the intron
Summary of the SnRNP s Roles in Splicing U1 binds the exon1-intron border U2 with the aid of U2AF binds the lariat junction sequence in the intron U6, U5 and U4 are a preassembled trimer which associate with U1 and U2 to bring the exons into alignment
Splicing and SnRNPs U6 associates with the 5 end ot the intron by base pairing U6 binds to U2 U5 associates with the last nucleotide of the exon1 and the first of exon2 U1 is released U4 is bound to U6 transiently until U6 is needed in the splicing reaction
Splicing and SnRNPs U4 is released and U5 binds stably to the 5 and 3 splice sites U2 and U6 catalyze the transesterification reaction forming the lariat and ligating the exon junctions
U6 and U5 Crosslinks to the pre-mrna
U2 U6 and U5 RNA interactions
The Spliceosome Cycle
3 Splice site selection Specific RNA binding proteins including the SR class of factors determine the selection of the 3 splice site
The yeast commitment complex A branchpoint bridging protein BBP binds to a protein in the U1 SnRNP at the 5 end of the intron and to Mud2p at the 3 end of the intron Thus these additional proteins may play a important initial role in setting up the spliceosome in yeast
The yeast commitment complex
Alternative Splicing: Immunoglobulin genes
Alternative Splicing: Sex determination in Drosophila
Group I Introns self splice Tetrahymena 26S rrna precursor intron can be removed in vitro with no protein addition to the RNA The reaction requires a guanine nucleotide on the 5 splice site, adding G to the 5 end of the intron and releasing the first exon The first exon attacks the 3 splice site ligating the two exons together and releasing the linear intron
Tetrahymena 26S rrna self splicing
What happens to the intron?
trna Splicing
Mechanism of trna splicing The intron is removed by a membrane bound endonuclease leaving a 2-3 phosphate group The 5 end of the 3 half of the molecule is phosphorylated by a kinase and the end is further modified by ligase to add an AMP molecule The two half-molecules are ligated through a 3-5 phosphodiester linkage Finally the 2 phosphate group is removed and a mature trna is formed
Mechanism of trna splicing
Precursor mrnas are capped Soon after polymerase clearance of the promoter a CTD bound enzyme guanylylmethyltransferase adds an inverted GDP molecule to the 5 end of the transcript
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Functions of Caps Protect mrna from degradation Enhance translation of the mrna Enhance the nuclear export of the mrna Enhance the efficiency of splicing
Poly AMP addition to the 3 end of the mrna Poly (A) polymerase adds approximately 250 AMP residues to the transcript Poly (A) increases the lifetime of the mrna and its ability to be translated A specific recognition sequence AAUAAA occurs approximately 20 bases before the poly (A) site
Poly (A) consensus sequence
Cleavage and poly (A) addition
Figure 15.26
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