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 5¹ end, an A at the branch point, a pyrimidine rich track between the branch point and the 3¹ end, and an AG at the 3¹ end.

 

Steps in the splicing process:

·  Splicing is thought to occur via two biochemical reactions and predicts that the intron has a 3¹OH, the phosphate between the exons comes from the 3¹ exon, the splicing intermediate has a branch nucleotide having 2¹ and 3¹ phosphodiester bonds, and the branch includes the 5¹ 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 3¹ phosphates after each nucleotide) and RNase P1 (leaves 5¹ 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 5¹ end of intron 1  and digestions with RNase T1 shows that the 5¹ end of intron 1 is part of the branch structure (F14.10).

·  The branch point is part of a conserved sequence close to the 3¹ 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 5¹ splice site (5¹ss) since certain mutations in the splice site are not spliced, but splicing is rescued by compensatory mutations in U1 for many (but not all) 5¹ss mutations (F14.15, F14.16).

b)  U6 snRNP base pairs with the 5¹ss (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 5¹ss through the action of Prp28 (part of the U5 snRNP) and ATP; When U6 binds the 5¹ss, U4 is released and U6 base pairs with U2 (F14.29).

·  The 3¹ss is an AG that is typically 18bp-40bp downstream from the branch point; Slu7 is required for selecting the proper 3¹ss  since depleting splicing extracts of Slu7 results in inappropriate 3¹ss selection (F14.30); U2AF, which binds to the polypyrimidine tract and the AG, is also required for 3¹ss selection.

·  Commitment refers to a state in which an intron is obliged to be spliced; SC35 is an ³SR¹ 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 5¹ss near the BP and the 3¹ss (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 3¹ss; 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).