RNA Splicing

Processing of Primary RNATranscripts into mRNA

Possible post-transcriptionalcontrols on gene expression.

Only a few of these controls arelikely to be used for any onegene.

Amount of DNA in the genomes of various organisms.

The Relationship Between Gene Size and mRNA Size

Species Average exon number Average gene length (kb) Average mRNA length (kb)

Hemophilus influenzae 1 1.0 1.0Methanococcus jannaschii 1 1.0 1.0

S. cerevisiae 1 1.6 1.6Filamentous fungi 3 1.5 1.5C. elegans 4 4.0 3.0D. Melanogaster 4 11.3 2.7Chicken 9 13.9 2.4Mammals 7 16.6 2.2------------------------------------------------------------------------SOURCE: Based on B. Lewin, Genes 5, Table 2-2. Oxford University Press.------------------------------------------------------------------------

Synthesis of a primary RNA transcript (an mRNA precursor) by RNA polymerase II.

This diagram starts with apolymerase that has justbegun synthesizing anRNA chain.

Recognition of a poly-Aaddition signal in thegrowing RNA transcriptcauses the chain to becleaved and thenpolyadenylated as shown.

In yeasts the polymeraseterminates its RNAsynthesis almostimmediately thereafter, butin higher eukaryotes itoften continuestranscription for thousandsof nucleotides.

The reactions that cap the 5' end of each RNA molecule synthesized by RNA polymerase II.

The final cap contains a novel 5'-to-5' linkagebetween the positively charged 7-methyl G residueand the 5' end of the RNA transcript. Polymerase Iand III transcripts are not capped. The indicatedreaction occurs almost immediately followinginitiation of each RNA chain. The letter N is usedhere to represent any one of the four ribonucleotides,although the nucleotide that starts an RNA chain isusually a purine (an A or a G). (After A.J. Shatkin,Bioessays 7:275-277, 1987. © ICSU Press.)

The first two reactions are catalyzed by a cappingenzyme that associates with the phosphorylatedCTD of RNA polymerase II shortly aftertranscription initiation. Two differentmethyltransferases catalyze reactions 3 and 4. S-adenosylmethionine (S-Ado-Met) is the source of themethyl (CH3) group for the two methylation steps;the guanylate (G) is methylated first, then the 2’hydroxyl of the first one or two nucleotides (N) inthe transcript. [See S. Venkatesan and B. Moss, 1982,Proc. Nat’l. Acad. Sci. USA 79:304.]

Capping enzyme:Phosphatase+Guanyl transferase

Methyl-transferases

Structure of the 5’ methylated cap of eukaryotic mRNA.

The distinguishing chemical features are the 5’ -5’ linkage of 7-methylguanylate to the initialnucleotide of the mRNA molecule and themethyl group on the 2’ hydroxyl of the ribose ofthe first nucleotide (base 1). Both these featuresoccur in all animal cells and in cells of higherplants; yeasts lack the methyl group on base 1.The ribose of the second nucleotide (base 2) alsois methylated in vertebrates. [See A. J. Shatkin,1976, Cell 9:645.]

Model for cleavage and polyadenylation ofpre-mRNAs in mammalian cells.

Cleavage-and-polyadenylation specificity factor (CPSF)binds to an upstream AAUAAA polyadenylation signal.

CStF interacts with a downstream GU- or U-richsequence and with bound CPSF, forming a loop in theRNA; binding of CFI and CFII help stabilize the complex.

Binding of poly(A) polymerase (PAP) then stimulatescleavage at a poly(A) site, which usually is 10 – 35nucleotides 3’ of the upstream polyadenylation signal.

The cleavage factors are released, as is the downstreamRNA cleavage product, which is rapidly degraded.Bound PAP then adds ≈12 A residues at a slow rate tothe 3’-hydroxyl group generated by the cleavagereaction.

Binding of poly(A)-binding protein II (PABII) to theinitial short poly(A) tail accelerates the rate of additionby PAP. After 200 – 250 A residues have been added,PABII signals PAP to stop polymerization.

Overview of RNA processing in eukaryotes using the β-globin gene as anexample.

The β-globin gene contains three protein-coding exons (red) and two interveningnoncoding introns (blue). The intronsinterrupt the protein-coding sequencebetween the codons for amino acids 31and 32 and 105 and 106. Transcription ofthis and many other genes starts slightlyupstream of the 5’ exon and extendsdownstream of the 3’ exon, resulting innoncoding regions (gray) at the ends ofthe primary transcript. These regions,referred to as untranslated regions (UTRs),are retained during processing. The 5’ 7-methylguanylate cap (m7Gppp; greendot) is added during formation of theprimary RNA transcript, which extendsbeyond the poly(A) site. After cleavage atthe poly(A) site and addition of multipleA residues to the 3’ end, splicing removesthe introns and joins the exons. The smallnumbers refer to positions in the 147-aasequence of β -globin.

Early evidence for the existence of introns in eukaryotic genes.

The evidence was provided by the "R-looptechnique," in which a base-pairedcomplex between mRNA and DNAmolecules is visualized in the electronmicroscope. An unusually abundantmRNA molecule, such as β-globin mRNAor ovalbumin mRNA, is readily purifiedfrom the specialized cells that produce it.When this single-stranded mRNApreparation is annealed in a suitablesolvent to a cloned double-stranded DNAmolecule containing the gene that encodesthe mRNA, the RNA can displace a DNAstrand wherever the two sequences matchand form regions of RNA-DNA helix.Regions of DNA where no match to themRNA sequence is possible are clearlyvisible as large loops of double-strandedDNA. Each of these loops (numbered 1 to6) represents an intron in the genesequence.

Consensus sequences for RNA splicing in higher eukaryotes.

The sequence given is that for the RNA chain; the nearly invariant GU and AGdinucleotides at either end of the intron sequence are highlighted in red, as is theconserved A at the branch point. The numbers below the nucleotides representpercent conservation.

Splicing of exons in pre-mRNA occurs viatwo transesterification reactions.

In the first reaction, the ester bondbetween the 5’ phosphorus of theintron and the 3’ oxygen (red) ofexon 1 is exchanged for an esterbond with the 2’ oxygen (dark blue)of the branch-site A residue. In thesecond reaction, the ester bondbetween the 5’ phosphorus of exon 2and the 3’ oxygen (light blue) of theintron is exchanged for an esterbond with the 3’ oxygen of exon 1,releasing the intron as a lariatstructure and joining the two exons.Arrows show where the activatedhydroxyl oxygens react withphosphorus atoms.

Structure of the branched RNA chain that formsduring nuclear RNA splicing.

The nucleotide shown in yellow is theA nucleotide at the branch site. Thebranch is formed in step 1 of thesplicing reaction, when the 5' end ofthe intron sequence couplescovalently to the 2'-OH ribose groupof the A nucleotide, which is locatedabout 30 nucleotides from the 3' endof the intron sequence. The branchedchain remains in the final excisedintron sequence and is responsiblefor its lariat form.

Analysis of RNA products formed in an in vitro splicing reaction

A nuclear extract from HeLa cells was incubatedwith a 497-nucleotide radiolabeled RNA (bottom)that contained portions of two exons (orange andtan) from human β-globin mRNA separated by a130-nucleotide intron (blue). After incubation forvarious times, the RNA was purified and subjectedto electrophoresis and autoradiography, along withRNA markers (lane M). The number of nucleotidesin the various species is indicated. Much of theslower-migrating starting RNA (497) was correctlyspliced, yielding a 367-nucleotide product. Theexcised intron (130*) migrated slower than expectedbased on its molecular weight, indicating that it isnot a linear molecule. Likewise, one of the reactionintermediates (339*) exhibited an anomalously slowelectrophoretic mobility. Additional analysisindicated that in both cases the intron had a lariatstructure resulting in the slow mobility. The 252**band, an aberrant product of the in vitro reaction, isgreatly reduced in reactions in which the RNA iscapped. [From B. Ruskin et al., 1984, Cell 38:317;photograph courtesy of Michael R. Green. See alsoR. A. Padgett et al., 1984, Science 225:898.]

The spliceosomal splicing cycle.

The splicing snRNPs (U1, U2, U4,U5, and U6) associate with the pre-mRNA and with each other in anordered sequence to form thespliceosome. Although ATPhydrolysis is not required for thetransesterification reactions, it isthought to provide the energynecessary for rearrangements of thespliceosome structure that occurduring the cycle. The branch-pointA in pre-mRNA is indicated inboldface. [See S. W. Ruby and J.Abelson, 1991, Trends Genet. 7:79;adapted from M. J. Moore et al.,1993, in R. Gesteland and J. Atkins,eds., The RNA World, Cold SpringHarbor Press, pp. 303-357.]

Diagram of interactions between pre-mRNA, U1 snRNA, and U2 snRNAearly in the splicing process.

The 5’ region of U1 snRNA initially base-pairs with nucleotides at the 5’ end of the intron (blue)and 3’ end of the 5’ exon (dark red) of the pre-mRNA; U2 snRNA base-pairs with a sequencethat includes the branch-point A, although this residue is not base-paired. The yeast branch-point sequence is shown here. Secondary structures in the snRNAs that are not altered duringsplicing are shown in diagrammatic line form. The purple rectangles represent sequences thatbind snRNP proteins recognized by anti-Sm antibodies. For unknown reasons, antisera frompatients with the autoimmune disease systemic lupus erythematosus (SLE) contain theseantibodies. Such antisera have been useful in characterizing components of the splicing reaction.[See E. J. Sontheimer and J. A. Steitz, 1993, Science 262:1989; adapted from M. J. Moore et al., 1993,in R. Gesteland and J. Atkins, eds., The RNA World, Cold Spring Harbor Press, pp. 303-357.]

The RNAcomponents ofsnRNPsare essential formRNA splicing

The two known classes of self-splicing intron sequences.The group I intron sequencesbind a free G nucleotide to aspecific site to initiatesplicing, while the group IIintron sequences use aspecially reactive Anucleotide in the intronsequence itself for the samepurpose. The twomechanisms have beendrawn in a way thatemphasizes their similarities.Both are normally aided byproteins that speed up thereaction, but the catalysis isnevertheless mediated by theRNA in the intron sequence.The mechanism used bygroup II intron sequencesforms a lariat and resemblesthe pathway catalyzed bythe spliceosome. (After T.R.Cech, Cell 44:207-210, 1986. ©Cell Press.)

Major Points1. Primary eukaryote RNA transcripts are processed by 5’ Capping, 3’polyA and internal intron removal by RNA splicing2. Many post-transcriptional steps can be regulated to form mRNA3. 5’ cap influences protein translation and 3’polyA tail effects stability4. Exon coding seq. are interrupted by intron seq. that must be spliced out of primary transcripts to form mature mRNA5. Intron removal involves self-splicing RNA seq. and splicesomes : protein/RNA complexes (RNP’s) containing special small U RNA’s6. Splicing occurs via 2 trans-esterification reactions and involve an intermediate branched RNA formed by linking 5’Phos of the intron to an A residue near the 3’ end of the intron via a 2’ OH7. Two classes of self-splicing introns: Group I ( uses G-OH) and Group II which uses the internal A residue to form a lariat & branch