Chapter 13 RNA splicing The chemistry of RNA splicing The spliceosome machinery Splicing pathways Alternative splicing Exon shuffling RNA editing mRNA transport

The chemistry of RNA splicing

Sequences within the RNA determine where splicing occurs

The intron is removed in a form called a lariat as the flanking exons are joined

Fig 13-5 The structure of the three-way junction formed during the splicing reaction

The splicing reactions have no net gain in the no. of chemical bonds The splicing reactions have no net gain in the no. of chemical bonds. Yet, a large number of ATP is consumed, not for the chemistry, but to properly assemble and operate the slicing machinery. What ensures the slicing only goes forward? Increase in entropy Excised intron is quickly degraded

Box 13-1 Adenovirus and the discovery of splicing Map of the human adenovirus-2 genome Human adenovirus is a DNA virus which serve as a model for studying eukaryotic gene regulation. Same promoter and the tripartate leader sequence for all the transcripts which are resulted from alternative splicing

R-loop mapping of the adenovirus-2 late messenger RNAs.

Exons from different RNA molecules can be fused by trans-splicing Fig 13-6 trans-splicing Although generally rare, trans-spicing occurs in almost all the mRNAs of trypanosomes (錐體蟲) eg. Nematode worms (C. elegans)

The spliceosome machinery RNA splicing is carried out by a large complex called the spliceosome Spliceosome comprises about 150 proteins and 5 RNAs and is similar in size with ribosomes. Many functions of spliceosome are carried out by RNAs rather by proteins. Five RNAs (U1, U2, U4, U5 and U6) are collectively called small nuclear RNAs (snRNAs) with the size in the range of 100-300 bp long and are complexed to small nuclear ribonuclear proteins (snRNP) The makeup of spliceosome varies at different stages of the splicing reaction.

Fig 13-7 some RNA-RNA hybrids formed during the splicing reaction

Structure of spliceosomal protein-RNA complex: U1A binds hairpin II of U1 snRNA

Splicing pathways Assembly, rearrangements, and catalysis within the spliceosome: the splicing pathway

Fig 13-8 steps of the spliceosome-mediated splicing reaction. Early (E) complex Fig 13-8 steps of the spliceosome-mediated splicing reaction. The A complex The B complex U1 snRNP replaced by U6 snRNP Active site formed U4 released, U2 forming RNA-RNA hybrids with U6 Lariat initially still bound with tri-snRNP. Soon the lariat degrades, leaving the snRNPs to be recycled.

Self-splicing introns reveal that RNA can catalyze RNA splicing class abundance mechanism Catalytic machinery Nuclear pre-mRNA Very common; used for most eukaryotic genes Two transestirification reactions; branch site A Major spliceosome Group II introns Rare; some eukaryotic genes from organelles and prokaryotes Same as pre-mRNA RNA enzyme encoded by intron (ribozyme) Group I introns Rare; nuclear rRNA in some eukaryotes, organelle genes, and a few prokaryotic genes Branch site G Same as group II

Group I introns release a linear intron rather than a lariat Fig 13-9 group I and group II introns Group I introns release a linear intron rather than a lariat

Fig 13-10 Proposed folding of the RNA catalytic regions for splicing of group II introns and pre-mRNAs.

Group I intron use a free G nucleoside or nucleotide Group I intron use a free G nucleoside or nucleotide. This G species is bound by the RNA and its 3’OH group is presented to the 5’ splice site. Structure of group I intron includes a binding pocket that will accommodate any guanine nucleoside and nucleotide. Internal guide sequence that base-pairs with the 5’ splice site sequence.

Box 13-1 group I introns can be converted into true ribozymes.

How does the spliceosome find the splice sites reliably? The average exon in only some 150 nt long, whereas the average intron is about 3,000 nt long. Thus, the exons must be identified within a vast ocean of intronic sequences.

Fig 13-12 Errors produced by mistakes in splice-site selection.

To avoid exon-skipping: co-transcriptional loading of spliceosome components onto the splice sites.(refer to 12-20) To avoid pseudo splice-site selection:

Fig 13-12 SR (serine-arginine rich) proteins recruit spliceosome components to the 5’ and 3’ splice sites. ESE: exonic splicing enhancer

A small group of introns are spliced by an alternative spliceosome composed of a different set of snRNPs Fig 13-13 The AT-AC (minor) spliceosome catalyzed splicing. This minor spliceosome has distinct splice site sequences but same splicing chemistry.

Alternative Splicing Single genes can produce multiple products by alternative splicing By alternative splicing multiple proteins can be produced from a single gene. These different proteins are called isoforms. They can have similar functions, distinct functions, or even antagonistic functions.

Fig 13-14 Alternative splicing in the troponin T gene

Fig 13-15 Five ways to splice a RNA

Fig 13-16 constitutive alternative splicing: monkey virus SV40 T-antigen 5’SST: 5’ splice site used to generate the large T mRNA 5’sst: 5’ splice site used to generate the small T mRNA

Several mechanisms exist to ensure mutually exclusive splicing Steric hindrance Combinations of major and minor splice sites 3. Nonsense-mediated decay

The curious case of the Drosophila Dscam gene: Mutually exclusive splicing on a grand scale

Dscam Down syndrome cell-adhesion molecule

Alternative splicing is regulated by activators and repressors Fig 13-22 Regulated alternative splicing Proteins that regulate splicing bind to specific sites called exonic (intronic) splicing enhancers (ESE/ISE) or silencers (ESS/ISS)

Fig 13-23 mammalian splicing repressor hnRNPI

Fig 13-25 a cascade of alternative splicing events determines the sex of a fly. Sxl: splicing repressor Tra: splicing activator

Exon Shuffling Exons are shuffled by recombination to produce genes encoding new proteins Why introns are present in all organisms except bacteria??? Introns early model: due to selection pressure to speed chromosome replication and cell division Introns late model: due to a transposome like mechanism Why have the introns been retained in (higher) eukaryotes?? The advantages of exon shuffling

Fig 13-26 Exons encode protein domains.

Fig 13-27 Genes made up of parts of other genes.

Fig 13-28 Accumulation, loss, and reshuffling of domains during the evolution of the family of chromatin modifying enzymes.

Evidences of exon shuffling The boundaries between exons/introns often coincide with boundaries between domains. Some proteins (eg. immunoglobulin) have repeating units, which might be due to gene duplications. Related exons are sometimes found in unrelated genes.

RNA Editing Site-specific deamination Guide RNA-directed uridine insertion or deletion (often in trypanosomes and mitochondria).

Human apolipoprotein gene Fig 13-29 RNA editing by deamination:

ADAR: adenosine deaminase acting on RNA

Fig 13-26 RNA-mediated editing by guide RNA mediated U insertion in trypanosome coxII gene.

mRNA Transport Once processed (capped, spliced, polyadenylated), mRNA is packaged and exported from the nucleus into the cytoplasm for translation How are RNA selection and transport achieved?

Fig 13-27 Transport of mRNAs out of the nucleus is an active process (export is though nuclear pore complex with the size exclusion of 50kd) SR protein or proteins that recognize exon-exon boundaries indicate a mature mRNA. Proteins that binds to introns (eg hnRNPs) indicate a RNA that needs to to retained.