Eukaryotic mrna is covalently processed in three ways prior to export from the nucleus:

Transcription

1 RNA Processing

2 Eukaryotic mrna is covalently processed in three ways prior to export from the nucleus: Transcripts are capped at their 5 end with a methylated guanosine nucleotide. Introns are removed by splicing. 3 ends are cleaved and extended with a poly(a) tail. All three processing events are intimately linked with transcription.

3 Cotranscriptional processing of nuclear pre-mrna The binding of processing factors is regulated by phosphorylation of specific amino acids in the CTD (serines at position 2 and 5 in the heptapeptide repeat ) Deletion of the RNA polymerase II CTD inhibits capping, splicing, and poly(a) cleavage.

4 Addition of the 5-7-methylguanosine cap The cap protects mrna from degradation and enhances the efficiency of splicing, nuclear export, and translation. A distinguishing chemical feature of the cap is the 5 5 linkage of 7- methylguanosine to the initial nucleotide of the mrna (m 7 GpppN).

5 The cap is added in three main steps: RNA triphosphatase cleaves the γ- phosphate at the 5 end of the growing RNA, leaving a diphosphate. RNA guanylyltransferase adds the capping GMP from GTP to the diphosphate at the end of the RNA, forming the 5 5 triphosphate linkage Methyltransferases transfer a methyl group to the N-7 of the capping guanosine and to the 2 -hydroxyl group of the ribose of the first and the second nucleotide (vertebrates).

6 Termination and polyadenylation Termination of transcription genes occurs at various positions rather than at a single site, and no consensus termination sequence has been identified. These regions are recognized by polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CstF).

7 Cleavage requires two additional complexes, mammalian cleavage factor I and II (CFIm, CFIIm). The mrna is cleaved while it is still being synthesized. After cleavage and release of the mrna, the transcript is polyadenylated at the 3 end. The poly(a) tail is added by poly(a) polymerase and enhances mrna stability and translation efficiency. Most eukaryotic mrnas have a chain of A residues about 100 to 250 nt long.

8 The poly(a) tails are coated with sequence-specific poly(a)-binding proteins. In the nucleus, PABPN1 increases the processivity of poly(a) polymerase. In the cytoplasm, PABPC functions in the initiation of translation and the regulation of mrna decay.

9 The remaining transcript still associated with RNA polymerase II is attacked by the Xrn2 exonuclease that chases after the polymerase. This exonuclease triggers dissociation of RNA polymerase from the DNA template and termination of transcription.

10 pre-mrna Introns are sequences that remain physically separated after excision. May contain transcriptional regulatory elements. May code for small nucleolar RNAs and micrornas. Exons are sequences that are ligated together after excision.

11 RNA splicing RNA splicing is the process by which introns are removed from a primary RNA transcript at precisely defined splice sites and the ends of the remaining RNA are rejoined to form a continuous mrna, ribosomal RNA (rrna), or transfer RNA (trna).

12 Major classes of introns are distinguished by their structure and mechanisms of splicing Self-splicing group I introns Self-splicing group II introns Large autocatalytic RNAs distinguished by their structure and mechanisms of splicing. Both types of introns have mobile members that have evolved mechanisms for inserting into intronless genes. trna introns Spliceosomal introns in nuclear pre-mrna

13 Group I & Group II introns

14 RIBOZYMES Group I & Group II introns RNA molecules with catalytic activity. Naturally occurring ribozymes can be autocatalytic, which lead to their own modification, or they can be true enzymes. Large ribozymes Vary in size from a few hundred to 3000 nucleotides. Cleave RNA to generate 3 -OH termini. RNA components of:

15 Group I introns Widely distributed in the mitochondrial, chloroplast, and nuclear genomes of eukaryotes. Nuclear group I introns are limited to rrna, whereas in organelles they are found in rrna, trna, and mrna. In animals, group I introns have only been found so far in the mitochondrial genomes of one sea anemone and a coral. Require an external G cofactor for splicing.

16 Two steps splicing reaction, involving two transesterification reactions Transesterification is the process in which phosphodiester linkages are broken and new ones are formed. 1. The RNA forms a complex threedimensional folding pattern which brings the two exons close together 2. Attack by an external guanine on the 5 splice site, adding the G to the 5 end of the intron and releasing the first exon. 3. The first exon attacks the 3 splice site, ligating the two exons together and releasing the linear intron which is then degraded.

17 Group II introns Less common than group I introns. Found in the mitochondrial and chloroplast, genomes of certain protists, fungi, algae, and plants. The 5 splice junction (GUGYG, where Y stands for any pyrimidine, C or U) and the 3 splice junction (AY) of group II introns are conserved. The RNA folds into a conserved structure that forms an active site containing catalytically essential Mg 2+ ions.

18 Splicing consists of two transesterification reactions and require an internal bulged A First transesterification reaction Attack by the 2 -OH of an internal bulged A on the 5 splice site. Release of the first exon. Formation of a lariat structure with a 2 5 phosphodiester bond.

19 Second transesterification reaction The first exon attacks the 3 splice site. The two exons are ligated together. The lariat intron is released and degraded.

20 Although many group I & II introns can self-splice in vitro, many, if not all, require proteins in vivo to fold into the catalytically active structure. Proteins required for splicing are either encoded by the introns themselves or by other genes of the host organism.

21 Nuclear trna introns

22 Some nuclear trna genes contain an intron Presence or absence of an intron defines two classes of eukaryotic nuclear trna genes. In humans, only trna Tyr and trna Leu contain introns. Nuclear trna splicing involves two main reactions: 1. Cleavage: The intron-containing pre-trnas are cleaved by an endoribonuclease at the 5 and 3 boundaries of the intron. 2. Joining: The paired trna halves are joined by ligase.

23 Spliceosomal introns in nuclear pre-mrna

24 Spliceosomal introns in nuclear pre-mrna Basic mechanisms is the same as self-splicing group II introns. Pre-mRNA introns do not themselves fold into conserved secondary or tertiary structures. The three-dimensional structure that is required for splicing is generated in a RNA protein complex termed the spliceosome.

25 Components of the spliceosome The spliceosome is the largest and most complex molecular machine known at present It contains five uracil-rich small nuclear RNA (snrna)- protein complexes termed snrnps (U1, U2, U4, U5, U6) and more than 200 proteins. The splicing machinery is recruited to intron-containing transcripts co-transcriptionally.

26 The border between introns and exons are marked by specific nucleotide sequence within the pre-mrna. The most highly conserved sequences are: - GU in the 5 splice site - AG in the 3 splice site - The A at the branch point site following by a - Polypyrimidine tract (Py tract)

27 Splicing pathway Formation of the E (early) or commitment complex U1 snrnp binds to the conserved 5 GU splice site. The 65 kd subunit of U2 auxiliary factor (U2AF) binds to the polypyrimidine tract. The 35 kda subunit of U2AF binds to the 3 AG splice site. The U2AF subunits interacts with branch point binding protein (BBP) and helps the protein to bind to the branch site This arrangement of proteins and RNA is called the early complex

28 Splicing pathway Formation of the A complex or pre-spliceosome U2 snrnp binds to the branch site region, aided by U2AF and displacing BBP. The base pairing between U2 snrnp and the branch site exclude the A residue with the result of a single nucleotide bulge. This A residue is thus unpaired and available to react with the 5 splice site.

29 Formation of the B complex Splicing pathway The A complex is joined by the U4/U6/U5 tri-snrnp.

30 Splicing pathway Formation of the C complex or spliceosome U1 leaves the complex and U6 replaces it at the 5 splice site. U4 is released from the complex allowing U6 to interact with U2. This arrangement, called C complex, produces the active site

31 Splicing pathway Formation of the active site juxtaposes the 5 splice site of the pre-mrna and the branch site, facilitating the first transesterification reaction. The second reaction, between the 5 and 3 splice site, is aided by the U5 snrnp, which helps to bring the two exons together. Spliceosome is disassembled. The lariat intron is degraded.

32 Protein factors that help mediate splicing Dynamic remodeling of the spliceosome requires the DEXH/D box RNA helicases. (DEXH/D motif: Asp-Glu-X-His/Asp, where X represents any amino acid). The recruitment of splicing factors is controlled by activating and inhibitory splice regulatory proteins, which can bind to exonic splicing enhancers and silencers (ESEs and ESSs, respectively), as well as to intronic splicing enhancers and silencers (ISEs and ISSs, respectively)

33 Protein factors that help mediate splicing Example: SC35 and ASF/SF2, members of the highly conserved serine/arginine (SR)-rich family of splicing factors have a dual role in stimulating constitutive and regulated splicing.

34 Alternative splicing

35 Alternative splicing A typical human or mouse gene contains 8-10 exons which can be joined in different arrangements by alternative splicing. Splicing of most exons is constitutive (they are always spliced or included in the final mature mrna). Splicing of some exons is regulated (they are either included or excluded from the mature mrna). At least 74% of human genes are alternatively spliced.

36 Effects of alternative splicing on gene expression Multiple alternative splicing positions in pre-mrnas gives rise to a family of related proteins from a single gene. Alternative splicing of 5 or 3 untranslated regions affects elements that regulate translation, stability, or mrna localization. Insertion of premature termination codons leads to nonsense-mediated decay. Regulation of gene expression by alternative splicing is important in many cellular and developmental processes, including sex determination, apoptosis, axon guidance, cell excitation, and cell contraction.consequently, errors in splicing regulation have been implicated in a number of different diseases.

37 Alternative splicing in mammalian heart development Alternative splicing of Ca 2+ /calmodulin-dependent kinase II (CaMKII ) has been shown to play a critical role in mammalian heart development. CaMKIIδ is a major intracellular mediator of Ca 2+ action in cells. Within the heart, it functions as a regulator of physical contraction of the heart muscle. The phosphorylation of key Ca 2+ -handling proteins is regulated, in part, by CaMKIIδ.

38 Alternative splicing in mammalian heart development In the developing fetal heart, the two cardiac isoforms (CaMKIIδB and CaMKIIδC) and one neuronal isoform (CaMIIδA) are expressed. Between 1 and 2 months of age, CaMKIIδ undergoes an alternative splicing transition, switching to the expression of the cardiac isoforms only. The different isoforms exhibit similar catalytic and regulatory activity. However, they differ completely in their intracellular localization Neuronal isoform ( A) is targeted to the transverse tubules of the sarcolemmal membranes. Cardiac isoform ( B) is targeted to the nucleus. Cardiac isoform ( C) is located in the cytoplasm of muscle cells. Inappropriate expression of the neuronal isoform ( A) leads to defects in heart development and function.

39 Trans-splicing The exon from one pre-mrna joins to an exon from another pre-mrna. A rare event. Occurs in special situations in organisms as diverse as flatworms, the protist Euglena gracilis, plant organelles, nematodes, and Drosophila.

40 Trans-splicing Three major types Discontinuous group II trans-splicing Spliced leader trans-splicing trna trans-splicing

41 Discontinuous group II trans-splicing First discovered in plant chloroplast genomes. Coding sequences are joined from separate transcripts to form a complete opening reading frame. Example: Trans-splicing events in NADH dehydrogenase 1 (nad1) pre-mrna

42 Spliced leader trans-splicing First discovered in African trypanosomes. A short sequence is added to the 5 termini of mrnas. The leader exon functions to: provide a 5 cap structure. resolve polycistronic transcripts into individual capped RNAs. enhance mrna translational efficiency.

43 trna trans-splicing In Nanoarchaeum equitans, nine genes encode separate trna halves which are then joined by trans-splicing.