Table 1201

Class of Intron

Classes of Intron

Location of Genes

GT-AG (or GU-AG) introns AT-AC (or AU-AC) introns Group I introns

Group II introns

Group III introns Twintrons Pre-tRNA introns Archeal introns eukaryotic nucleus (common) eukaryotic nucleus (rare) organelles, prokaryotes (rare) rRNA

in lower eukaryotes organelles (of plants and fungi), some prokaryotes organelles organelles tRNA of eukaryotic nucleus archaebacterial tRNA and rRNA

The poly(A)-binding protein (PABP) stays associated with the mRNA and binds to the poly(A) tail. It is suspected that the PABP may bind both ends of the mRNA as it also appears to protect the cap from being cut off (see below). Whether the presence of the poly(A) tail and PABP make the mRNA more stable is doubtful; some stable mRNAs have very short tails. The poly(A) tail is required for translation. Certain mRNAs in early embryos are stored without a poly(A) tail and cannot be translated. When they are needed for translation, the poly(A) tail is added. Thus, mRNA may be stored.

Some mRNAs of bacteria are also polyadenylated. However, the role of the poly(A) tail is quite different in prokaryotes. In fact, the poly(A) tail triggers degradation of prokaryotic mRNA. The bacterial poly(A) polymerase is associated with the ribosomes and the tails are relatively short (10-40 bases). Addition of a poly(A) tail to mRNA in chloroplasts also promotes its degradation. This is another indication of the prokaryotic ancestry of these organelles (see Ch. 20).

Introns are removed and the exons forming the coding sequence are joined together by the spliceosome.

snRNA molecules recognize the ends of the intron as well as the future branch site.

Introns are Removed from RNA by Splicing

After capping and tailing, the next step in processing pre-mRNA is the splicing out of the introns. Splicing must be accurate to within a single base since a mistake would throw the whole coding sequence out of register and totally scramble the protein resulting from translation of the mRNA. The overall result of this cutting and pasting has been depicted in Figure 12.01. There are several classes of introns (Table 12.01). The most frequent class of intron in eukaryotic nuclear genes is the GT-AG (or GU-AG in RNA code) group of introns. We will therefore discuss these first, before surveying the other variants.

The splicing machinery is known as the spliceosome and consists of several proteins and some specialized, small RNA molecules found only in the nucleus (Fig. 12.08). Each small nuclear RNA (snRNA) plus its protein partners forms a small nuclear ribonucleoprotein (snRNP) or "snurp". There are five snRNPs—numbered from U1 to U6 with U3 missing! (U3 is actually a snoRNA found in the nucleolus—see below.)

The snRNAs of the snurps recognize three sites on the pre-mRNA. These are the 5' and 3' splice sites and the branch site. The vast majority of introns start with GU and end with AG. Recognition is due to base pairing between the snRNA and the

3' splice site Recognition site for splicing at the downstream or 3'-end of the intron 5' splice site Recognition site for splicing at the upstream or 5'-end of the intron branch site Site in the middle of an intron where branching occurs during splicing small nuclear ribonucleoprotein (snRNP) Complex of snRNA plus protein snurp snRNP or small nuclear ribonucleoprotein spliceosome Complex of proteins and small nuclear RNA molecules that removes introns during the processing of messenger RNA

Eukaryotic mRNA may be isolated by taking advantage of its poly(A) tail. Artificial strands of oligo(U) or oligo(dT) will base pair with poly(A) tracts. Generally, the oligo(U) or oligo(dT) is immobilized on a column and the mixture containing mRNA is poured through the column. The mRNA is trapped by binding of its poly(A) tail (Fig. 12.07). Other molecules, in particular non-coding RNA, pass through.

Mixture of RNA

Resin carrying oligo (dT)

Mixture of RNA

Resin carrying oligo (dT)

rRNA tRNA do not bind

FIGURE 12.07 Binding of Poly(A) Tail Allows Isolation of Eukaryotic mRNA

rRNA tRNA do not bind

FIGURE 12.07 Binding of Poly(A) Tail Allows Isolation of Eukaryotic mRNA

Oligo (dT) attached to a resin binds the mRNA molecules by base pairing with their poly(A) tails. Ribosomal RNA and transfer RNA molecules are not bound and exit the column. The mRNA is then eluted in purified form.

The spliceosome is made of several snRNA molecules plus accompanying proteins.

pre-mRNA. The protein part of the snurp supervises the cutting and joining reactions. This is shown in detail in Figure 12.09 for the U1 snurp which recognizes the 5' splice site. In the middle of the intron is a special adenine residue used as a branch site during splicing. The consensus recognition sequences for the 5' splice site, 3' splice site and branch site are as follows (residues in bold are most highly conserved as they are involved in the splicing mechanism):

3' splice site: 5'-YYYYYYNCAG fl -3' (Y = any pyrimidine)

branch site: 5'-UACUAAC-3'

The snurps assemble onto the pre-mRNA,so forming the spliceosome (Fig. 12.10). U1 recognizes the 5' splice site, U2 binds the branch site (Fig. 12.09B) and a protein oligo(dT) DNA strand consisting only of thymidine oligo(U) RNA strand consisting only of uridine U1 Snurp (snRNP) that recognizes the upstream splice site U2 Snurp (snRNP) that recognizes the branch site

FIGURE 12.08 Spliceosome Recognizes Intron/Exon Boundaries

The spliceosome consists of several ribonucleoproteins (U1 to U6), also known as "snurps", which are involved in splicing. These assemble at the splice sites at the intron/exon boundaries.

Boundary

3' splice site

5' splice site Boundary

Boundary

3' splice site

FIGURE 12.08 Spliceosome Recognizes Intron/Exon Boundaries

The spliceosome consists of several ribonucleoproteins (U1 to U6), also known as "snurps", which are involved in splicing. These assemble at the splice sites at the intron/exon boundaries.

5' splice site Boundary

A) BASE PAIRING OF U1 TO SPLICE SITE

,U1RNA

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