Introns Are Removed Exons Are Spliced Together

The mechanisms whereby introns are removed from the primary transcript in the nucleus, exons are ligated to form the mRNA molecule, and the mRNA molecule is transported to the cytoplasm are being elucidated. Four different splicing reaction mechanisms have been described. The one most frequently used in eukaryotic cells is described below. Although the sequences of nu-

Exon 1

Exon 2

^ Intron

Exon 1

Exon 2

^ Intron

Cap-

Cap-

Ligation of 3' end of exon end of exon 2

Intron is digested

Figure 37-11. The processing of the primary transcript to mRNA. In this hypothetical transcript, the 5' (left) end of the intron is cut (4-) and a lariat forms between the G at the 5' end of the intron and an A near the 3' end, in the consensus sequence UACUAAC. This sequence is called the branch site, and it is the 3' most A that forms the 5'-2' bond with the G. The 3' (right) end of the intron is then cut (4). This releases the lariat, which is digested, and exon 1 is joined to exon 2 at G residues.

cleotides in the introns of the various eukaryotic transcripts—and even those within a single transcript—are quite heterogeneous, there are reasonably conserved sequences at each of the two exon-intron (splice) junctions and at the branch site, which is located 20-40 nucleotides upstream from the 3' splice site (see consensus sequences in Figure 37-12). A special structure, the spliceosome, is involved in converting the primary transcript into mRNA. Spliceosomes consist of the pri mary transcript, five small nuclear RNAs (U1, U2, U5, U4, and U6) and more than 60 proteins. Collectively, these form a small nucleoprotein (snRNP) complex, sometimes called a "snurp." It is likely that this penta-snRNP spliceosome forms prior to interaction with mRNA precursors. Snurps are thought to position the RNA segments for the necessary splicing reactions. The splicing reaction starts with a cut at the junction of the 5' exon (donor or left) and intron (Figure 37-11). This

Figure 37-12. Consensus sequences at splice junctions. The 5' (donor or left) and 3' (acceptor or right) sequences are shown. Also shown is the yeast consensus sequence (UACUAAC) for the branch site. In mammalian cells, this consensus sequence is PyNPyPy-PuAPy, where Py is a pyrimidine, Pu is a purine, and N is any nucleotide. The branch site is located 20-40 nucleotides upstream from the 3' site.

Figure 37-12. Consensus sequences at splice junctions. The 5' (donor or left) and 3' (acceptor or right) sequences are shown. Also shown is the yeast consensus sequence (UACUAAC) for the branch site. In mammalian cells, this consensus sequence is PyNPyPy-PuAPy, where Py is a pyrimidine, Pu is a purine, and N is any nucleotide. The branch site is located 20-40 nucleotides upstream from the 3' site.

is accomplished by a nucleophilic attack by an adenylyl residue in the branch point sequence located just upstream from the 3' end of this intron. The free 5' terminal then forms a loop or lariat structure that is linked by an unusual 5'-2' phosphodiester bond to the reactive A in the PyNPyPyPuAPy branch site sequence (Figure 37-12). This adenylyl residue is typically located 28-37 nucleotides upstream from the 3' end of the intron being removed. The branch site identifies the 3' splice site. A second cut is made at the junction of the intron with the 3' exon (donor on right). In this second transesterification reaction, the 3' hydroxyl of the upstream exon attacks the 5' phosphate at the downstream exon-intron boundary, and the lariat structure containing the intron is released and hy-drolyzed. The 5' and 3' exons are ligated to form a continuous sequence.

The snRNAs and associated proteins are required for formation of the various structures and intermediates. U1 within the snRNP complex binds first by base pairing to the 5' exon-intron boundary. U2 within the snRNP complex then binds by base pairing to the branch site, and this exposes the nucleophilic A residue. U5/U4/U6 within the snRNP complex mediates an ATP-dependent protein-mediated unwinding that results in disruption of the base-paired U4-U6 complex with the release of U4. U6 is then able to interact first with U2, then with U1. These interactions serve to approximate the 5' splice site, the branch point with its reactive A, and the 3' splice site. This alignment is enhanced by U5. This process also results in the formation of the loop or lariat structure. The two ends are cleaved, probably by the U2-U6 within the snRNP complex. U6 is certainly essential, since yeasts deficient in this snRNA are not viable. It is important to note that RNA serves as the catalytic agent. This sequence is then repeated in genes containing multiple introns. In such cases, a definite pattern is followed for each gene, and the introns are not necessarily removed in se-quence—1, then 2, then 3, etc.

The relationship between hnRNA and the corresponding mature mRNA in eukaryotic cells is now apparent. The hnRNA molecules are the primary transcripts plus their early processed products, which, after the addition of caps and poly(A) tails and removal of the portion corresponding to the introns, are transported to the cytoplasm as mature mRNA molecules.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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