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

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

mRNA Capping RULE: Capping the 5’ end of mRNAs serves 2 purposes. Added Guanine 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 5’-5’ triphosphate CH3 Gppp + pppApNpNp… CH3 GpppApNpNp… + pp + p

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

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)

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.

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

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

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)

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.

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

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.

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

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

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

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

Processing t-RNA Cut Spliced out

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