RNA Processing Lecture Notes PDF

1. rRNA & tRNA processing - rRNA processing; Pulse-chase - tRNA processing; RNase E & RNase P 2. Capping and Polyadenylation: - Structure of mRNA caps -Functions of caps: stability, translation, transport -Structure and function of poly(A) tails -Cleavage and polyadenylation signals -Turnove...

1. rRNA & tRNA processing - rRNA processing; Pulse-chase - tRNA processing; RNase E & RNase P 2. Capping and Polyadenylation: - Structure of mRNA caps -Functions of caps: stability, translation, transport -Structure and function of poly(A) tails -Cleavage and polyadenylation signals -Turnover of poly(A)  Eukaryotic rRNA and tRNA Processing Like mRNAs, rRNAs and tRNAs must be processed. That is, pieces of the originally transcribed RNA must be removed before the RNA reaches the mature form. Unlike mRNAs, rRNAs and tRNAs contain extra RNA at the 5’ and 3’ ends that must be removed. In eukaryotes, the ribosomal RNAs that will eventually make up the ribosome are initially transcribed by pol I as a long 45S rRNA precursor (the exact length varies by species. First the 5’ spacer is removed, then 41S precursor is cleaved into the 20S and 32S precursors. The 32S is cut into the mature 5.8S and 28S rRNAs, and the 20S is trimmed into the 18S. The 5.8S and 28S rRNAs base pair in Fig. 16.2 the mature ribosome. 16-2  16.1 Ribosomal RNA Processing rRNA genes of both eukaryotes and bacteria are transcribed as larger precursors must be processed to yield rRNAs of mature size Several different rRNA molecules are embedded in a long, precursor and each must be cut out In eukaryotes, many copies of the DNA encoding the rRNA genes are clustered together. They are separated by NTSs (non-transcribed spacers). The Fig. 16.1 ribosomal RNAs are the most highly transcribed RNAs in the cell, and the gap between the rRNA genes and the NTSs are clearly visible by EM. The rRNA genes are concentrated to a distinct, electron dense region of ribosome assembly in the nucleus nucleolus called the nucleolus. nucleus 16-3 The ribosomal RNAs (rRNAs) are transcribed as a precursor that is processed into the individual components How does the intensity of the peaks change over time?  How can the order of rRNA processing established? (example of a pulse chase experiment) When a time-course of a biological process is to be investigated, a very useful experiment is a pulse-chase. rRNA is briefly labeled with radioactivity (3H-uridine, or 32P-ATP, the pulse). Then the radioactivity is chased using non-radioactive (cold) uridine or ATP, and the fate of the radioactivity incorporated into the rRNA is tracked over time. At right, the radioactivity initially incorporated into the 45S rRNA gradually becomes the 28S and 18S rRNA. Curr. Protoc. Cell Biol. 39:22.11.1-22.11.16. The order and specificity of rRNA processing is orchestrated by the small nucleolar ribonucleoproteins, or snoRNPs, which contain small nucleolar RNAs (snoRNAs). Conceptually, these are similar to the way that the snRNPs direct and order splicing in the spliceosome: they interact with the rRNA precursor and direct where processing will occur. It is also thought that assist the ribosomal RNA in reaching the correct conformation to make a functional ribosome; that is, they have RNA chaperone function.  Processing Bacterial rRNA Like in eukaryotes, the bacterial rRNA is first transcribed as a long precursor. One enzyme that is known to be very important in the cleavage of the precursor into the mature forms is RNAse III. The sequences flanking the 23S rRNA gene are predicted to create an extended hairpin, which is the recognition site for this enzyme. Note that the cleavage sites on either side of the hairpin are offset by 2 nucleotides (more on this later) 16.4 16-6  16.2 Transfer RNA Processing Transfer RNAs are made in all cells as overly long precursors (pre-tRNAs) – These must be processed by removing RNA at both ends Nuclei of eukaryotes contain precursors of a single tRNA In bacteria, precursor may contain one or more tRNA (like a tRNA “operon”). Like for bacterial rRNA processing, the enzyme that first cleaves this into individual tRNA precursors is RNase III. 16-7  RNase P Action The first step in tRNA processing is removal of the extra RNA at the 5’ end of the pre-tRNA, called the leader. This is performed in both prokaryotes and eukaryotes by an enzyme called RNAse P. This yields the correct, mature 5’ end of the pre-tRNA. The RNAse P enzyme was discovered to have two components, one protein and one RNA. Which component catalyzed the pre-tRNA cleavage? 16.5 16-8  The catalytic component of RNAse P is RNA Separation of the RNA and protein component of E. coli RNAse P resulted in a loss of cleavage activity. However, incubation of a pre-tRNA with the RNA from RNAse P (called the M1 RNA) with increasing magnesium concentration resulted in an RNA that could cleave the tRNA leader, confirming that this RNA was catalytic (i.e. an enzyme). This activity was specific for the pre-tRNA, as a pre-4.5S RNA was not cleaved. We now know that many RNAs required Mg++ to reach their native (correct) conformation. Fig. 16.6 This discovery of catalytic RNA won Sid Altman ( ) a Nobel prize (along with Tom Cech) in 1989.  Posttranscriptional Events II: Capping and Polyadenylation Two modifications to pre-mRNA apart from splicing: 1) Capping – a blocking nucleotide, m7Gppp is added to 5’ end 2) Polyadenylation – a string of AMPs is added to 3’ end 10  Cap Structure The m7G is linked by three phosphates to the next nucleotide: 1) The α phosphate, but not β or γ, of GTP is retained in the cap. 2) The α and β phosphates of ATP, that initiated RNA synthesis, are retained in the cap. 11 How are caps synthesized? From vaccinia and reoviruses (replicate in cytoplasm): a) RNA triphosphatase removes γ- phosphate b) Guanylyl transferase attaches GMP from GTP c) Methyl transferase transfers methyl group from SAM to N7of guanine d) Another methyl transferase uses another SAM to methylate the 2’- hydroxyl of the next nucleotide 12 When is cap added? in some viruses, such as cytoplasmic polyhedrosis virus, lack of SAM completely inhibits transcription. Implies that capping is very early, soon after first phosphodiester bond in the pre-mRNA. in vaccinia virus, transcription is not stopped by lack of SAM. adenovirus replicates in nucleus and takes advantage of cell’s capping machinery so likely simulates pre-mRNAs. Caps are added before chain length reaches 70nt. Generally accepted that capping occurs on mRNAs before chain length reaches 30nt. 13  Function of Caps 1) Protect mRNA from degradation 2) Enhance translation of mRNAs 3) Enhance transport of mRNAs from nucleus to cytoplasm 4) Enhance efficiency of splicing To show protection: Synthesized in vitro capped (m7GpppG), blocked (GpppG) or uncapped reovirus RNAs Injected RNAs into Xenopus oocytes and incubated 8h Purified viral RNAs and separated by glycerol gradient ultracentrifugation 14 Results: a) Prior to incubation, three size classes (s,m,l) of RNAs b) After incubation, all RNAs have some degradation, but uncapped suffers most Capped RNA Blocked RNA Uncapped RNA 15  Function #2: Translatability of Capped RNA Cap confers 297 fold stimulation of translation on mRNA with PolyA tail. 16  Transport of mRNA U1 snRNA is transcribed by Pol II and transcripts get m7G caps. Transcripts travel to cytoplasm, bind to proteins to form snRNPs, and their caps get trimethylated. They re-enter nucleus as active complex. U6 snRNA is transcribed by Pol III and stays uncapped, and remains in the nucleus. What happens if U1 gene is placed under control of Pol III promoter? Does snRNA stay in the nucleus if it doesn’t have a cap? U1 made by Pol III remained in the nucleus. This is consistent with the presence of the cap being important for RNA export to the cytoplasm. 17