Molecular Biology

Chapter 14 Outline

Intron discovery and boundaries:

∑ Genes contain introns that must be spliced out to form a mature mRNA (F14.2).

∑ Large nuclear RNAs called hnRNA are unspliced precursors to mRNAs; Introns are transcribed as shown by R-looping experiments (F14.3).

∑ Accurate splicing is essential for generating the correct protein sequence; Four consensus sequences are found in introns ≠ GU at the 5π end, an A at the branch point, a pyrimidine rich track between the branch point and the 3π end, and an AG at the 3π end.

Steps in the splicing process:

∑ Splicing is thought to occur via two biochemical reactions and predicts that the intron has a 3πOH, the phosphate between the exons comes from the 3π exon, the splicing intermediate has a branch nucleotide having 2π and 3π phosphodiester bonds, and the branch includes the 5π end of the intron (F14.4).

∑ Lariat intermediates run at anomalous sizes on acrylamide gels (F14.5); RNase T1 cleavage shows that the end of intron 1 has an OH (F14.7); ³²P labeling the C at the beginning of exon 2 and cleaving with RNase A shows that the resulting spliced product contains the ³²P (F14.8); Use of RNase T2 (leaves 3π phosphates after each nucleotide) and RNase P1 (leaves 5π phosphates after each nucleotide) shows that the intermediate (exon 2 + intron) and spliced intron contain a branched nucleotide having an anomalous charge (F14.9); Hybridizing an oligonucleotide to the 5π end of intron 1 and digestions with RNase T1 shows that the 5π end of intron 1 is part of the branch structure (F14.10).

∑ The branch point is part of a conserved sequence close to the 3π splice site that is required for splicing (F14.11).

Spliceosome structure:

∑ Splicing intermediates from yeast are in 40S particles dubbed spliceosomes; Human splicesosomes are 60S particles containing splicing intermediates and products (F14.13).

∑ Within the spliceosome are small nuclear RNAs (snRNAs) that recognize splicing signals; snRNAs U1, U2, U4, U5 and U6 are part of small nuclear ribonuclear proteins (snRNPs).

a) U1 snRNP is required for splicing and base pairs with the 5π splice site (5πss) since certain mutations in the splice site are not spliced, but splicing is rescued by compensatory mutations in U1 for many (but not all) 5πss mutations (F14.15, F14.16).

b) U6 snRNP base pairs with the 5πss (F14.17) and 4-thio-U crosslinking shows this interaction occurs by the time lariats have formed (F14.18); Psoralen crosslinking reveals that U6 also associates with the splicing substrate at the initial stage of splicing and with U2 later on (F14.19b).

c) U2 snRNP can base pair with sequence around the branch point; Splicing of a gene with mutant branch point sequences can be rescued by compensatory changes in U2, thus demonstrating that base pairing occurs (F14.20, F14.21); U2 also base pairs with U6 based on similar compensatory base pair change experiments.

d) U5 snRNP associates with the last base of exon 1 and the first base of exon 6 as demonstrated by crosslinking U5 with 4-thio-U labeled first base of exon 2 or last base of exon 1 and primer extending U5/premRNA, U5/E1 and U5/intron-E2 products (F14.22a, F14.23a, F14.23b, F14.23d, F14.24).

e) U4 base pairs with and sequesters U6, then dissociates after splicing has commenced, allowing U6 to bind U2 and form an active splicesosome (F14.26).

Spliceosome assembly and function:

∑ U1 snRNP is the first to add to the pre-mRNA (F14.27), then U2 adds to the pre-mRNA complex in an ATP dependent manner (F14.28); U6 displaces U1 from the 5πss through the action of Prp28 (part of the U5 snRNP) and ATP; When U6 binds the 5πss, U4 is released and U6 base pairs with U2 (F14.29).

∑ The 3πss is an AG that is typically 18bp-40bp downstream from the branch point; Slu7 is required for selecting the proper 3πss since depleting splicing extracts of Slu7 results in inappropriate 3πss selection (F14.30); U2AF, which binds to the polypyrimidine tract and the AG, is also required for 3πss selection.

∑ Commitment refers to a state in which an intron is obliged to be spliced; SC35 is an ≥SRπ RNA binding protein that promotes commitment as its affect on b-globin pre-mRNA can not be competed with excess competitor pre-mRNA (F14.31ab); Commitment occurs within 1 minute and is ATP independent (F14.31c); Other pre-mRNAs require different/multiple SR proteins for commitment (F14.32, F14.33).

∑ Yeast commitment was studied by identifying proteins that interacted with the U1 snRNP; Mud2p function in commitment requires U1 and a sequence near the branchpoint; Mud2p function also requires branchpoint bridging protein (BBP); Yeast two hybrid analysis (F14.34) showed that BBP bound U1 snRNP and Mud2p bound to BBP (F14.35); BBP also binds to the branchpoint, thus holding the 5πss near the BP and the 3πss (F14.36); Mud2p and BBP homologs are found in mammals (F14.36).

∑ Many genes are alternatively spliced to form different transcripts and proteins; Alternative splicing controls sex determination in Drosophila (F14.38); Tra and Tra2 are RNA binding proteins that bind ~300bp downstream of the female specific 3πss; Tra and Tra2 are necessary for commitment to female specific splicing of dsx and act in concert with SR proteins (F14.39).

Self-splicing RNAs;

∑ Some RNAs can splice themselves without a spliceosome, thus demonstrating RNA catalysis; Group I and Group II self splicing introns use different mechanisms; Splicing occurs via hydroxyl groups attacking phosphodiester bonds (F14.45); The linear intron continues to self-splice (F14.46); Group II splicing occurs through interactions similar to splicesosome dependent splicing (F14.26).