1. Processing of eukaryotic pre-mRNA Human dystrophin gene has 79 exons, spans over 2,300-Kb and requires over 16 hours to be transcribed! For primary transcripts containing multiple exons and introns, splicing occurs before transcription of the gene is complete--co- transcriptional splicing.

2. Sometimes methylated Sometimes methylated The cap is added after the nascent RNA molecules produced by RNA polymerase II reach a length of 25- 30 nucleotides. Guanylyltransferase is recruited and activated through binding to the Ser5-phosphorylated Pol II CTD. The methyl groups are derived from S- adenosylmethionine. Capping helps stabilize mRNA and enhances translation, splicing and export into the cytoplasm. Capping of the 5’ end of nascent RNA transcripts with m 7 G Existing in a single complex

3. Polyadenylation of mRNA at the 3’ end CPSF: cleavage and polyadenylation specificity factor. CStF: cleavage stimulatory factor. CFI & CFII: cleavage factor I & II. PAP: poly(A) polymerase. PABPII: poly(A)-binding protein II. Poly(A) tail stabilizes mRNA and enhances translation and export into the cytoplasm. RNA is cleaved 10~35-nt 3’ to A 2 UA 3. The binding of PAP prior to cleavage ensures that the free 3’ end generated is rapidly polyadenylated. PAP adds the first 12A residues to 3’-OH slowly. Binding of PABPII to the initial short poly(A) tail accelerates polyadenylation by PAP. The polyadenylation complex is associated with the CTD of Pol II following initiation.

4. Consensus sequences around 5’ and 3’ splice sites in vertebrate pre-mRNAs The central region of the intron, which may range from 40 bases to 500 kilobases in length, generally is unnecessary for splicing to occur.

5. Thalassemia: a group of inherited anemias characterized by defective synthesis of hemoglobin (O 2 -transporter with  2  2 subunits) is caused by mutations in  -globin gene splice sites Normal: 5’-CCTATTGGTCTATTTTCCACCCTTAGGCTGC-3’  -thalassemia:5’-CCTATTAGTCTATTTTCCACCCTTAGGCTGC-3’ Stop codon Normal 3’ end of intron A new 3’ splice site due to a G to A mutation, leading to aberrant splicing of  -globin gene.

6. The splicing reaction proceeds in two steps Step 1. Cleavage at the 5’ splice site and joining of the 5’ end of the intron to the branch point A within the intron, producing a lariat-like intermediate. Step 2. Cleavage at the 3’ splice site and simultaneous ligation of the exons, resulting in excision of the intron as a lariat-like structure. Two transesterification reactions: the number of phosphodiester bonds remains unchanged in either reaction. Two transesterification reactions: (1) The 5’ P of the intron is attacked by the 2’-OH of the branch site Adenosine, causing cleavage of a 3’, 5’-phosphodiester bond and formation of a 2, 5’- phosphodiester bond (not hydrolysis followed by ligation). (2) The newly formed 3’-OH of exon 1 attacks the 5’ P of exon 2, causing cleavage of a phosphodiester bond and formation of a new bond.

7. Model of spliceosome-mediated splicing of pre-mRNA Five snRNPs (U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles) containing 5 snRNAs (U1, U2, U4, U5 and U6 small nuclear RNAs, ranging from 107 to 210 nucleotides) and their associated proteins (6-10 per snRNP) assemble on the pre-mRNA to form the spliceosome. There are a total of ~100 proteins in the spliceosome, some of which are not associated with snRNPs. These non-snRNP proteins may contribute to the specificity of recognition of the splice sites by snRNPs and some of them contain RNA helicase activity to help the rearrangements of base pairing in snRNAs during the splicing cycle. U4 masks the catalytic activity of U6 in the U4/U6/U5 tri- snRNPs prior to the actual transesterification reactions. Massive rearrangements of base-pairing interactions among various snRNAs converts the spliceosome into a catalytically active form, which releases the U1 and then the U4 snRNPs and brings U2 and U6 together. RNA molecules play key roles in directing the alignment of splice sites (e.g. U1 and U2 base pairing with the pre-mRNA) and in carrying out the catalysis (a U2/U6 catalytic center).

8. Base pairing between pre-mRNA, U1 snRNA, and U2 snRNA early in the splicing cycle and experimental demonstration that the base pairing between U1 and the 5’ splice site in pre-mRNA is important

9. Cell type-specific splicing of fibronectin pre-mRNA in fibroblasts and hepatocytes: concept of alternative splicing Fibroblasts produce fibronection with exons EIIIA and EIIIB, which allow the protein to adhere to proteins in the fibroblast plasma membranes and enable fibroblasts to stick to the extracellular matrix. Hepatocytes produce fibronection without EIIIA and EIIIB, which circulates in the serum and is important during the formation of blood clots. The presence of multiple introns in many eukaryotic genes permits expression of multiple, related proteins from a single gene by means of alternative slicing, an important mechanism for the production of different forms of proteins, called isoforms, by different types of cells. Nearly 60% of all human genes are expressed as alternatively spliced mRNAs, leading to an expansion of the coding capacity of our genome.

10. Question: You are using Northern blotting to analyze two mRNA samples derived from fibroblasts and hepatocytes. What will you see if you use a probe made from exon EIIIB of the fibronectin gene? What about using a probe made from the exon next to EIIIB? Detection of alternative splicing by Northern blotting Northern blotting can be used to detect specific RNAs in complex mixtures. Southern blotting detects specific DNA fragments. Western blotting (immunoblotting) detects specific proteins with antibodies. RNA mixture Transfer solution

11. Exonic splicing enhancers (ESEs) and SR proteins contribute to exon definition and regulate alternative splicing The correct 5’ GU and 3’ AG splice sites are recognized by splicing factors on the basis of their proximity to exons. The exons contain exonic splicing enhancers (ESEs) that are binding sites for SR proteins. When bound to ESEs, the SR proteins interact with one another and promote the cooperative binding of the U1 snRNP to the 5’ splice site of the downstream intron, the 65- and 35-kD subunits of U2AF to the pyrimidine-rich region and 3’ AG splice site of the upstream intron. The bound U2AF also helps recruit U2 snRNP to the branch point. The resulting RNA- protein cross-exon recognition complex spans an exon and activates the correct splice sites for RNA splicing and this exon is retained in the final spliced mRNA. If U1 snRNP and/or U2AF are not recruited to the splice sites on each side of an exon (no formation of a stable cross-exon recognition complex), this exon will not be recognized and, instead, will be excised as part of the intron.

12. The RNA Pol II CTD is required for the coupling of transcription with mRNA capping, polyadenylation and splicing 1.The coupling allows the processing factors to present at high local concentrations when splice sites and poly(A) signals are transcribed by Pol II, enhancing the rate and specificity of RNA processing. 2.The association of splicing factors with phosphorylated CTD also stimulates Pol II elongation. Thus, a pre- mRNA is not synthesized unless the machinery for processing it is properly positioned.

13. Splicing mechanisms in group I and group II self-splicing introns and spliceosome-catalyzed splicing of pre-mRNA; discovery of self-splicing of ribosomal RNA in Tetrahymena Occurs in pre-rRNAs from Tetrahymena and mitochondria & chloroplast origins. The 1 st cleavage is carried out by an external cofactor guanosine (G, 3’-OH). The intron is released in a linear form. Group II self-splicing introns are found in mitochondria and chloroplast pre-mRNAs. The 1 st cleavage is carried out by the 2’-OH of A within the intron. The intron is released in the form of a lariat. Common to all: two transesterification reactions are involved.

14. Catalytic introns ----- RNA as enzymes ------ Ribozymes During evolution, there has been a transfer of catalytic power from the intron itself to other molecules such as snRNPs, which are specialized in carrying out splicing reactions. Introns nowadays are variable in size because they are no longer self- splicing and this increases the capacity of regulation. The similarity between these two suggests that the U snRNAs probably evolved from group II introns of endosymbiotic organelles.