(and some other important stuff) Chapter 13 RNA Splicing (and some other important stuff) 13 and 15 October, 2004

Overview Introns are removed after transcription - exons may comprise only a few percent of the primary transcript. The location of splicing is determined by splice site consensus sequences. The intron is released as a lariat structure. Splicing is carried out by a large ribonucleoprotein structure called the spliceosome. Spliceosome snRNPs use RNA base pairing to recognize splice sites. Spliceosome components assemble at the 5’ splice site (U1/U6), the Branch point (U2), and the 3’ splice site (U5). There are at lease two types of self-splicing introns. Splicing is highly regulated, including many instances of alternative splicing. Some RNAs are edited before translation. Polyadenylation is coupled to transport.

Primary transcript and spliced product hybridization suggest splicing.

Splicing

Splicing Consensus Sequences

Unusual Electrophoretic Behavior of Intron and Dependence on snRNPs

The Splicing Reaction

Structure of the Lariat Intermediate

Trans Splicing

snRNP-RNA Recognition

Splicosome Purification

Splicosome Assembly

Base pairing between U2 and branch point sequences is demonstrated by second-site suppressor studies.

4-thioU crosslinking demonstrates interactions between U6 and the 5’ splice site, U5 and the 3’ splice site.

The Spliceosome Cycle

The two-hybrid system demonstrates interactions between components of the commitment complex.

Self-Splicing Introns

Spliced and Self-splicing Introns

Alternative Splice Sites

Splicing Errors

Regulation of Alternative Splicing

Alternative Splicing

Alternative Splicing

Inhibition of Splicing

AT-AC Spliceosome

Conservation of Splicing Consenses

Exons encode protein domains.

Gene Assembly

Protein Evolution

RNA Editing

C to U deamination

Editing Mechanism

mRNA Transport

Purification of a poly-A binding protein that stimulates poly-A polymerase (PABII)

Model for polyadenylation

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