Cpsf

5'Q t IAAUAAA

signal Poly(A) site signal

CPSF, CStF, CFI, CFII

Pre-mRNA

Pre-mRNA

Cleavage

Cleavage

3'

ATP Slow pp. polyadenylation

AAAoh 3

AAAoh 3

PABII ■

V^. pp polyadenylation

V^. pp polyadenylation

AAUAAAhH A~1^A~1^A~1^tA~200oh 3

p products in in vitro processing reactions performed with nuclear extracts of HeLa cells.

Early sequencing of cDNA clones from animal cells showed that nearly all mRNAs contain the sequence AAUAAA 10-35 nucleotides upstream from the poly(A) tail. Polyadenylation of RNA transcripts is virtually eliminated when the corresponding sequence in the template DNA is mutated to any other sequence except one encoding a closely related sequence (AUUAAA). The unprocessed RNA transcripts produced from such mutant templates do not accumulate in nuclei, but are rapidly degraded. Further mutagenesis studies revealed that a second signal downstream from the cleavage site is required for efficient cleavage and polyadenylation of most pre-mRNAs in animal cells. This downstream signal is not a specific sequence but rather a GU-rich or simply a U-rich region within «50 nucleotides of the cleavage site.

Identification and purification of the proteins required for cleavage and polyadenylation of pre-mRNA have led to the model shown in Figure 12-4. According to this model, a 360-kDa cleavage and polyadenylation specificity factor (CPSF), composed of four different polypeptides, first forms an unstable complex with the upstream AAUAAA poly(A) signal. Then at least three additional proteins bind to the CPSF-RNA complex: a 200-kDa heterotrimer called cleavage stimulatory factor (CStF), which interacts with the G/U-rich sequence; a 150-kDa heterotrimer called cleavage factor I (CFI); and a second, poorly characterized cleavage factor (CFII). Finally, a poly(A) polymerase (PAP) binds to the complex before cleavage can occur. This requirement for PAP binding links cleavage and polyadenylation, so that the free 3' end generated is rapidly polyadenylated.

Assembly of the large, multiprotein cleavage/polyadenyl-ation complex around the AU-rich poly(A) signal in a pre-mRNA is analogous in many ways to formation of the transcription-preinitiation complex at the AT-rich TATA box of a template DNA molecule (see Figure 11-27). In both cases, multiprotein complexes assemble cooperatively through a network of specific protein-nucleic acid and protein-protein interactions.

Following cleavage at the poly(A) site, polyadenylation proceeds in two phases. Addition of the first 12 or so A residues occurs slowly, followed by rapid addition of up to 200-250 more A residues. The rapid phase requires the binding of multiple copies of a poly(A)-binding protein containing the RRM motif. This protein is designated PABPII to distinguish it from the poly(A)-binding protein present in the cytoplasm. PABPII binds to the short A tail initially added by PAP, stimulating polymerization of additional A residues by PAP (see Figure 12-4). PABPII is also responsible for signaling poly(A) polymerase to terminate polymerization when the poly(A) tail reaches a length of 200-250 residues, although the mechanism for controlling the length of the tail is not yet understood.

Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions

During formation of a mature, functional mRNA, the introns are removed and exons are spliced together. For short transcription units, RNA splicing usually follows cleavage and polyadenylation of the 3' end of the primary transcript, as depicted in Figure 12-2. However, for long transcription units

M EXPERIMENTAL FIGURE 12-5 RNA-DNA hybridization studies show that introns are spliced out during pre-mRNA processing. Electron microscopy of an RNA-DNA hybrid between adenovirus DNA and the mRNA encoding hexon, a major viral protein, reveals DNA segments (introns) that are absent from the hexon mRNA. (a) Diagram of the FcoRI A fragment of adenovirus DNA, which extends from the left end of the genome to just before the end of the final exon of the hexon gene. The gene consists of three short exons and one long («3.5 kb) exon separated by three introns of «1, 2.5, and 9 kb. (b) Electron micrograph (left) and schematic drawing (right) of hybrid between an fcoRI A fragment and hexon mRNA. The loops marked A, B, and C correspond to the introns indicated in (a). Since these intron sequences in the viral genomic DNA are not present in mature hexon mRNA, they loop out between the exon sequences that hybridize to their complementary sequences in the mRNA. [Micrograph from S. M. Berget et al., 1977, Proc. Nat'l. Acad. Sci. USA 74:3171; courtesy of P A. Sharp.]

5' splice site

Branch point

Pyrimidine-rich region (=15 b)

3' splice site

5' splice site

Branch point

Pyrimidine-rich region (=15 b)

3' splice site

5' Exon

Intron 1 1

3' Exon

Pre-mRNA

A/C A G

G U A/G A G U C U A/G A C/U

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