rRNA & tRNA Processing

rRNA & tRNA Processing

• Eukaryotic and bacterial rRNA genes are transcribed into larger precursors which must be processed into mature rRNAs.
• Several different rRNA molecules are embedded in a precursor, and each must be cut out.
• In eukaryotes, DNA encoding rRNA genes are clustered together and separated by non-transcribed spacers (NTSs).
• Ribosomal RNAs are the most highly transcribed RNAs in the cell. The gap between rRNA genes and NTSs are clearly visible by electron microscopy.
• rRNA genes are concentrated in the nucleolus, a distinct, electron-dense region of ribosome assembly in the nucleus.
• Ribosomal RNAs are transcribed as a precursor and processed into individual components.
• Eukaryotic 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 the 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 the mature ribosome.
• In a pulse-chase experiment rRNA is briefly labeled with radioactivity, then the radioactivity is chased using non-radioactive uridine or ATP. The fate of the radioactivity is then tracked.
• Radioactivity initially incorporated into the 45S rRNA gradually becomes the 28S and 18S rRNA over time.
• The order and specificity of rRNA processing is orchestrated by small nucleolar ribonucleoproteins (snoRNPs), which contain small nucleolar RNAs (snoRNAs)
• Bacterial rRNA is transcribed as a long precursor; one enzyme is important in cleavage of the precursor into mature forms is RNAse III.
• Sequences flanking the 23S rRNA gene create an extended hairpin, which is the recognition site for this enzyme. The cleavage sites on either side of the hairpin are offset by 2 nucleotides.
• Transfer RNAs are made in all cells as overly-long precursors (pre-tRNAs which must be processed by removing RNA at both ends.
• Bacterial precursors might contain one or more tRNA (like a tRNA “operon”). RNase III cleaves this into individual tRNA precursors.
• The first step in tRNA processing is removal of the extra RNA at the 5’ end of the pre-tRNA, called the leader, and is performed by RNAse P, which yields the mature 5’ end of the pre-tRNA.
• The RNAse P enzyme has two components, one protein and one RNA.
• The RNA component is the catalytic component of RNAse P.

Capping and Polyadenylation

• Capping is the process of adding a blocking nucleotide, m7Gppp, to the 5’ end of pre-mRNA.
• Polyadenylation is the process of adding a string of AMPs to the 3’ end
• The m7G is linked by three phosphates to the next nucleotide. The α phosphate, but not β or γ, of GTP is retained in the cap. The α and β phosphates of ATP initiated RNA synthesis and are retained in the cap.
• In cap synthesis RNA triphosphatase removes γ phosphate, then guanylyl transferase attaches GMP from GTP.
• Methyl transferase transfers a methyl group from SAM to N7 of guanine. Another methyl transferase uses another SAM to methylate the 2’-hydroxyl of the next nucleotide
• Some viruses, like cytoplasmic polyhedrosis virus, lack SAM, and capping occurs very early, soon after the first phosphodiester bond in the pre-mRNA.
• Adenovirus replicates in the nucleus and likely simulates pre-mRNAs. Caps are added before a chain length reaches 70nt. Capping occurs on mRNAs before chain length reaches 30nt.
• The functions of caps include protecting the mRNA from degradation, enhancing translation of mRNAs, enhancing transport of mRNAs from the nucleus to the cytoplasm, and enhancing the efficiency of splicing.
• Capped mRNAs are more stable, and uncapped mRNAs suffer the most degradation.
• A Cap confers 297 fold stimulation of translation on mRNA with PolyA tail.
• U1 snRNA is transcribed by Pol II and gets m7G caps, travels to the cytoplasm, binds to proteins to form snRNPs, and the caps get trimethylated. They re-enter the nucleus as an active complex.
• U6 snRNA is transcribed by Pol III and stays uncapped, and remains in the nucleus.
• U1 made by Pol III remained in the nucleus, which is consistent with the presence of a cap being important for RNA export to the cytoplasm.