RNA Processing

1. RNA Processing • Capping • Polyadenylation • Introns vs exons • Splicing • Genomic vs cDNA • Ribosomal RNA processing • t-RNA processing

2. Prokaryotes vs Eukaryotes PRO: All three classes of RNA are synthesized by one polymerase. EU: There are 3 major RNA polymerases. Pol 1 synthesizes rRNA; Pol 2 synthesizes mRNA; Pol 3 synthesizes tRNA. PRO: mRNA undergoes hardly any posttranscriptional processing. It is translated as it is synthesized. EU: mRNA is capped, polyadenylated, spliced PRO: mRNA contains no introns EU: mRNA contains intervening sequences (introns) that are removed during processing

3. Added Guanine 5’-5’ triphosphate mRNA Capping CH3 RULE: Capping the 5’ end of mRNAs serves 2 purposes. First, the cap protects the mRNA from 5’-exonuclease activity. Second, the cap interacts with eIF-2, a translation initiation factor required to position the mRNA on the ribosome CH3 Gppp + pppApNpNp… CH3 GpppApNpNp… + pp + p

4. Polyadenylation In eukaryotes, mRNA is polyadenylated by an enzyme system that cuts the RNA 30 bs downstream from an AAUAAA, then adds A to the 3’ end at the cleavage site Thus, poly A is not coded in the DNA, but is added after transcription

5. Dialogue Q: How should one picture a typical mammalian gene? A: Mammalian genes have both introns and exons. Only the exons encode information that will appear in a protein. Q: What are introns? A: Introns appear in unprocessed mRNA. The term is a shortened version of the words “intervening sequences” Q: How do exons differ from introns? A: One could say that the typical mammalian gene has 7 or 8 exons in a length of about 15 kb. The exons are short, (100-200 bp) whereas introns are large (>1000 bp)

6. Q: What happens to introns? A: During nuclear processing, the introns are spliced out and exons are joined together in a linear continuum Q: How is this accomplished? A: Cells have mechanism that recognize introns. The most common is a spliceosome that recognizes the boundaries of intron-exon junctions and knows were to cleave and splice Q: What is involved in the recognition? A: Small RNA molecules that work with spliceosomes, The RNA hybridizes to the residues at the splice junctions and tells the enzyme where to cut.

7. Stages in the Life of a Typical mRNA DNA Transcription via RNA Pol II Primary Transcript Capping and polyadenylation Capped-polyadenylated mRNA Splicing Mature mRNA

8. Intron I is generally quite large in mammalian mRNAs Chick Ovalbumin Introns form loops that help with their excision and splicing 1,872 nucleotides (24.3%) represented as exons

9. Dialogue on mRNA Splicing What can you tell me about splicing? Ans: Splicing occurs in two steps. First the junction at the 5’-end of the intron is broken. This is called the 5’-splice site Then what happens? Ans: The -OH on the 3’ end of the liberated exon becomes a nucleophile and attacks the 3’-splice site of the intron. This is the second step. The two exons are now joined. What happens to the intron? Ans: The intron is set free. Because a 2’-OH on an adenosine caused the initial cleavage, there is a loop in the intron (called a lariat)

10. Does this happen automatically Ans: Sometimes. Some RNAs are capable of self-splicing. Most of the time the splicing occurs through a large complex called a spliceosome. What is a spliceosome? Ans: A spliceosome is a giant 50-60S particle composed of splicing proteins, pre-mRNAs and small nuclear RNA proteins or “SNURPS”. What do the snRNPs do? Ans: The 5’-end of some of the snRNPs complements bases at the splice junctions. The snRNPs probably locate the exon-intron boundaries which tend to be constant for all eukaryote mRNAs. There are about 6 of them U1-U6. Not all functions are known.

11. OH2’ pApA pApA pApA pGpU pGpU pGpU pApG pApG pApG pGp pGp pGp A C U1-snRNP O OH O Exon 1 Exon 2 -OH3’ Spliced exons Intron with lariat Exon 2 Exon 1 Adenosine Intron

12. Ribosomal RNA Q: Is ribosomal RNA processed the same way as mRNA? A: No Q: How is it different? A: In bacteria, r-RNA is not spliced, it is only cut. All processing is done with a special class of RNAases Q: What about eukaryotes? A: Eukaryotes employ basically the same mechanism, but they also can engage in self-splicing Q: What is self-splicing? A: Self-splicing implies that a pre-rRNA can carry out its own splicing without the need of a spliceosome.

13. Q: Does this mean the RNA is acting as its own nuclease? A: Yes. It is called a ribozyme in recognition of its catalytic activity. Q: How does self-splicing work? A: In self-splicing 3’-OH group on the donor exon is primed by an attack by GMP, GDP, or GTP. Q: Then what? A: As before, the freed -OH attacks the phosphate at the 3’ end and forms a new linkages that joins to two segments

14. Ribosomal RNA Primary transcript 2920 300 150 200 1700 5’ 3’ F P P III III III E III RNase: P F Primary processing Pre-5S rRNA Pre 16S rRNA Pre 23S rRNA 5’ 3’ D D RNase: M16 M16 M23 M23 M5 5S rRNA 16S rRNA tRNA(s) 23S rRNA tRNA(s) Number of bases 5’ 3’ 1541 2904 120 Bacteria rRNA 16S 23S 5S 4S 4S Secondary processing

15. Stages in the Life of a Eukaryotic Ribosomal RNA 45S RNA 5’ 3’ 18S 5.8S 28S Heavily methylated RNase III, RNase P DNA (300 randomly repeated copies of rRNA genes in the genome that are transcribed via RNA pol I and processed in the nucleolus) spacers Primary Transcript Methylation at 110 sites Methylated Transcript tRNA and 5S sequences are not part of the 45S transcript

16. Self-splicing pre-rRNA Any guanine nucleotide (GMP, GDP,GTP) sets it off

17. Cut Spliced out Processing t-RNA

19. Amino acid attachment site methyladenosine 2’-methylguanosine dihydrouridine isopentenyladenosine Anticodon loop  pseudouridine Note: mature t-RNA has the highest number of odd bases See p345 in Strategies