【正文】 Gene Splicing ? The introns are rare in prokaryotic structural genes ? The introns are unmon in lower eukaryote (yeast), 239 introns in ~6000 genes, only one intron / polypeptide ? The introns are abundant in higher eukaryotes (lacking introns are histons and interferons) ? Unexpressed sequences constitute ~80% of a typical vertebrate structural gene Errors produced by mistakes in splicesite selection Mechanisms prevent splicing error ? Cotranscriptional loading process ? SR proteins recruit spliceosome ponents to the 5’ and 3’ splice sites ? SR protein = Serine Arginine rich protein ? ESE = exonic splicing enhancers ? SR protein regulates alternative splicing Alternative splicing ? Alternative splicing occurs in all metazoa and is especially prevalent in vertebrate Five ways to splice an RNA Regulated alternative splicing Different signals in the premRNA and different proteins cause spliceosomes to form in particular positions to give alternative splicing 7 6 5 7 5 6 5 7 Fas premRNA APOPTOSIS Alternative splicing can generate mRNAs encoding proteins with different, even opposite functions (programmed cell death) Fas ligand Soluble Fas (membrane) Fas Fas ligand (membrane associated) (+) () Alternative possibilities for 4 exons leave a total number of possible mRNA variations at 38,016. The protein variants are important for wiring of the nervous system and for immune response. Drosophila Dscam gene contains thousands of possible splice variants Cis and TransSplicing Cis: Splicing in single RNA Trans: Splicing in two different RNAs Yshaped excised introns (cis: lariat) Occur in C. elegance and higher eukaryotes but it does in only a few mRNAs and at a very low level premRNA splicing transmRNA splicing spliced leader Same splicing mechanism is employed in transsplicing Spliced leader contains the cap structure! RNA editing ? RNA editing is the process of changing the sequence of RNA after transcription. ? In some RNAs, as much as 55% of the nucleotide sequence is not encoded in the (primary) gene, but is added after transcription. ? Examples: mitochondrial genes in Trypanosomes （錐蟲） ? Can add, delete or change nucleotides by editing Two mechanisms mediate editing ? Guide RNAdirected uridine insertion or deletion ? Sitespecific deamination Insertion and deletion of nucleotides by editing ? Uses a guide RNA (in 20S RNP = editosome) that is encoded elsewhere in the genome ? Part of the guide RNA is plementary to the mRNA in vicinity of editing Trypanosomal RNA editing pathways. (a) Insertion. (b) Deletion. Mammalian example of editing The C is converted to U in intestine by a specific deaminating enzyme, not by a guide RNA. Cutting and Trimming RNA ? Can use endonucleases to cut at specific sites within a longer precursor RNA ? Can use exonucleases to trim back from the new ends to make the mature product ? This general process is seen in prokaryotes and eukaryotes for all types of RNA The posttranscriptional processing of E. coli rRNA. RNase III cuts in stems of stemloops 16S rRNA 23S rRNA RNase III No apparent primary sequence specificity perhaps RNase III recognizes a particular stem structure. Eukaryotic rRNA Processing ? The primary rRNA transcript (~7500nt, 45S RNA) contains 18S, and 28S ? Methylation occur mostly in rRNA sequence 80%: O2methylribose, 20%: bases (A or G) ? peudouridine 95 U in rRNA in human are converted to Y’s may contribute rRNA tertiary stability Transfer RNA Processing ? Cloverleaf structure ? CCA: amino acid binding site ? Anticodon ? ~60 tRNA genes in E. coli A schematic diagram of the tRNA cloverleaf secondary structure. Endo and exonucleases to generate ends of tRNA ? Endonuclease RNase P cleaves to generate the 5’ end. ? Endonuclease RNase F cleaves 3 nucleotides pa