PTM

Messenger RNAs Undergo 5 Capping and Addition of a 3 Tail

• In prokaryotes, most primary mRNA transcripts are translated without further modification. Indeed, protein synthesis usually begins before transcription is complete. In eukaryotes, however, mRNAs are synthesized in the cell nucleus, whereas translation occurs in the cytosol. Eukaryotic mRNA transcripts can therefore undergo extensive posttranscriptional processing while still in the nucleus.

Eukaryotic mRNAs Have 5 Caps

• Eukaryotic mRNAs have a cap structure consisting of a 7-methylguanosine (m7G) residue joined to the transcript’s initial (5’) nucleotide via a 5’–5’ triphosphate bridge.

• The cap, which adds to the growing transcript when it is ~30 nt long, identifies the eukaryotic translation start site

• Capping involves several enzymatic reactions: (1) the removal of the leading phosphate group from the mRNA’s 5’ terminal triphosphate group by an RNA triphosphatase; (2) the guanylylation of the mRNA by capping enzyme, which requires GTP and yields the 5’–5’ triphosphate bridge and PPi ; and (3) the methylation of guanine by guanine-7-methyltransferase, in which the methyl group is supplied by S-adenosylmethionine (SAM).

• In addition, the cap may be O2’-methylated at the first and second nucleotides of the transcript by a SAM-requiring 2’- O-methyltransferase. Capping enzyme binds to RNAP II’s phosphorylated CTD, and hence it appears that capping marks the completion of RNAP II’s switch from transcription initiation to elongation. Capped mRNAs are resistant to 5’-exonucleolytic degradation.

FIG. 26-20 Structure of the 5¿ cap of eukaryotic mRNAs. A cap may be O2’-methylated at the transcript’s leading nucleoside (the predominant cap in multicellular organisms), at its first two nucleosides, or atneither of those positions (the predominant cap in unicellular eukaryotes). If the first nucleosideis adenosine (it is usually a urine), it may also be N6-methylated.

Eukaryotic mRNAs Have Poly(A) Tails

• Mature eukaryotic mRNAs, however, have well-defined 3’ ends terminating in poly(A) tails of ~250 nt (~80 nt in yeast). The poly(A) tails are enzymatically appended to the primary transcripts in two reactions:

• A transcript is cleaved 15 to 25 nt past a highly conserved AAUAAA sequence and less than 50 ntbefore a less-conserved U-rich or G + U–rich sequence. The precision of this cleavage reaction has apparently eliminated the need for accurate transcription termination.

• Nevertheless, the identity of the endonucleasethat cleaves the RNA is uncertain although cleavage factors I and II (CFI and CFII) are required for this process.

• The poly(A) tail is subsequently generated from ATP through the stepwise action of poly(A) polymerase (PAP), a template-independent RNA polymerase that elongates an mRNA primer with a free 3’-OH group. PAP is activated by cleavage and polyadenylation specificity factor (CPSF) when the latter protein recognizes the AAUAAA sequence.

• Once the poly(A) tail has grown to ~10 residues, the AAUAAA sequence is no longer required for further chain elongation

• CPSF binds to the phosphorylated RNAP II CTD; deleting the CTD inhibits polyadenylation

• poly(A) binding protein II (PAB II) determine length of poly A tail.

• cleaved transcript is polyadenylated before it can dissociate and be digested by cellular nucleases

Splicing Removes Introns from Eukaryotic Genes

• The primary transcripts, also called pre-mRNAs or heterogeneous nuclear RNAs (hnRNAs), are variable in length and are much larger (~2000 to > 20,000 nt)

• intervening sequences (introns),

• flanking expressed sequences (exons)

• In humans, the number of introns in a gene varies from none to 364 (in the ~2400-kb gene encoding the 34,350-residue muscle protein titin, the largest known gene and single-chain protein, with intron lengths ranging from ~65 to ~800,000 nt (in the gene encoding the muscle protein dystrophin; and averaging ~3500 nt (exons, in contrast, have lengths that average ~150 nt and range up to 17,106 nt in the gene encoding titin).

• The introns from corresponding genes in two vertebrate species rarely vary in number and position, but often differ extensively in length and sequence so as to bear little resemblance to one another.

• The production of a translation-competent eukaryotic mRNA begins with the transcription of the entire gene, including its introns (Fig. 26-22). Capping occurs soon after initiation, and splicing commences during the elongation phase of transcription. The mature mRNA emerges only after splicing is complete and the RNA has been polyadenylated. The mRNA is then transported to the cytosol, where the ribosomes are located, for translation into protein.

Exons Are Spliced in a Two-Stage Reaction

• GU at the intron’s 5’ boundary and an invariant AG at its 3’ boundary.

• These sequences are necessary and sufficient to define a splice junction

• The splicing reaction occurs via two transesterification reactions

• A 2’,5’-phosphodiester bond forms between an intron adenosine residue and the intron’s 5’-terminal phosphate group. The 5’ exon is thereby released and the intron assumes a novel lariat structure (so called because of its shape). The adenosine at the lariat branch point is typically located in a conserved sequence 20 to 50 residues upstream of the 3’splice site. Mutations that change this branch point A residue abolish splicing at that site.

• The 5’ exon’s free 3 ’ OH group displaces the 3 ’ end of the intron, forming a phosphodiester bond with the 5 ’ -terminal phosphate of the 3 ’ exonand yielding the spliced product. The intron is thereby eliminated in its lariat form with a free 3 ’ -OH group. Mutations that alter the conserved AG at the 3 ’ splice junction block this second step, although they do not interfere with lariat formation. The lariat is eventually debranched(linearized) and, in vivo, is rapidly degraded.

• Exon skipping

• This occurs, at least in part, because splicing occurs cotranscriptionally

• Splicing Is Mediated by snRNPs in the Spliceosome

• The eukaryotic nucleus contains numerous copies of highly conserved 60- to 300-nt RNAs called small nuclear RNAs (snRNAs), which form protein complexes termed small nuclear ribonucleoproteins (snRNPs

• U1-snRNA (which is so named because of its high uridine content), is partially complementary to the consensus sequence of 5’splice junctions. Apparently, U1-snRNP recognizes the 5 ’splice junction. Other snRNPsthat participate in splicing are U2-snRNP, U4–U6-snRNP (in which the U4- and U6-snRNAs associate via base pairing), and U5-snRNP.

• Splicing takes place in an ~60S particle dubbed the spliceosome. The spliceosome brings together a pre-mRNA, the snRNPs, and a variety of premRNA binding proteins. The spliceosome, which consists of 5 RNAs and ~150 polypeptides, is comparable in size and complexity to the ribosome

• All four snRNPs involved in mRNA splicing contain the same so-called snRNP core protein, which consists of seven Sm proteins, named B, D1, D2, D3, E, F, and G proteins. These proteins collectively bind to a conserved AAUUUGUGG sequence known as the Sm RNA motif, which occurs in U1-, U2-, U4-, and U5-snRNAs.

• Mammalian U1-snRNP consists of U1-snRNA and ten proteins, namely, the seven Sm proteins that are common to all U-snRNPs as well as three that are specific to U1-snRNP: U1-70K, U1-A, and U1-C.

• The predicted secondary structure of the 165-nt U1-snRNA contains five double-helical stems, four of which come together at a four-way junction.

• U1-70K and U1-A bind directly to RNA stem–loops (SL) 1 and 2, respectively, whereas U1-C is bound by other proteins

• Fortuitously, the 5’ ends of two neighboring U1-snRNAs in the crystal form a double-helical segment, which serves as a model for how the 5 ’ end of U1-snRNA base-pairs with the 5 ’ splice site of the pre-mRNA in the spliceosome. The zinc finger domain of U1-C interacts with this double helix and presumably stabilizes it. This is consistent with the observation that mutants of U1-C in this region cannot initiate spliceosome formation.

Alternative mRNA Splicing Yields Multiple Proteins from a Single Gene

• The expression of numerous cellular genes is modulated by the selection of alternative splice sites. Thus, genes containing multiple exons may give rise to transcripts containing mutually exclusive exons. In effect, certain exons in one type of cell may be introns in another. For example, a single rat gene encodes seven tissue-specific variants of the muscle protein -tropomyosin through the selection of alternative splice sites