RNA Processing and snoRNP Complexes

Pre-rRNA Processing

• Pre-rRNAs are processed in the nucleolus by processing complexes containing small nucleolar RNPs (snoRNP).
• In yeast, the process involves the pre-rRNA, 170 nonribosomal proteins, 70 snoRNPs, and 78 ribosomal proteins.
• Each snoRNP catalyzes the modification of one or two rRNA nucleotides (nt).

snoRNPs

• Each snoRNP combines a snoRNA of 60 to 300 nt with four or five proteins/enzymes.
• 10 to 21 nt of a snoRNA is complementary to a sequence on the rRNA – identifying a base for modification.
• One snoRNP type produces pseudouridine.

tRNA Processing

• Cells have 40 to 50 tRNAs, which are processed from longer precursors by removing 5’ and 3’ nt.
• Rnase P, an RNP with catalytic RNA, cleaves the 5’ end.
• Rnase D cleaves the 3’ end.
• In eukaryotes, some tRNA have introns that need to be excised.
• Specific nt are methylated, deaminated, or reduced to control its interaction with proteins and the ribosome.
• The 3’ CCA trinucleotide, to which the respective amino acid is attached, is mostly added by tRNA nucleotidyltransferase.

Degradation of Cellular mRNAs

• RNA lifetime is a method of gene regulation.
• Half-lives of mRNA vary from seconds to hours.
• Typical half-life of vertebrate mRNA is ~3 h.
• The pool of each mRNA turns over ~10 fold per cell generation.
• Bacterial mRNA half-lives are only ~1.5 min.
• Degradation occurs via ribonucleases.
• In eukaryotes, transcription terminates when an endonuclease cleaves the nascent RNA from Pol II.
• A poly(A) tail is added to the 3’ end by polyadenylate polymerase.

Alternative Splicing and Poly(A) Site Choice

• Alternative splicing and alternative poly(A) site choice in eukaryotes create many different transcripts from a single gene.
• Primary transcripts of tRNAs, rRNAs, and miRNAs are extensively processed by endonucleolytic cleavage and chemical modification aided by snoRNPs.

RNA Degradation

• RNA degradation is tightly regulated.
• In bacteria, endonucleases fragment mRNA for destruction by exonucleases.
• In eukaryotes, mRNAs are decapped and the poly(A) tail shortened before degradation.
• The exosome is a supramolecular complex of exo- and endonucleases involved in many steps of eukaryotic RNA decay.

Mutations in SMN1 and Splicing

• Mutations in SMN1 create too little functional protein, resulting in neuromuscular degeneration.
• The drug nusinersen, an 18 nt oligomer, corrects the splicing of SMN2, creating active SMN2 protein.

Alternative Splicing of the Calcitonin Gene

• The combination of alternative splicing and different poly(A) sites greatly increases the variety of proteins in higher eukaryotes.
• The calcitonin gene transcript in rats has two poly(A) sites, one used in the brain and the other in the thyroid.

Posttranscriptional Modifications

• Bases in tRNA and pre-rRNA are chemically modified by posttranscriptional enzymatic reactions, creating 96 derivatives: 81 in tRNAs, 30 in rRNAs.

Bacterial Ribosomal RNAs (rRNA)

• In bacteria, a single 30S RNA precursor of 6500 nt is cleaved by RNases III, P, and E to create the 16S, 23S, and 5S ribosomal RNAs (rRNA) – as well as some tRNAs.
• The 16S rRNA includes a pseudouridine and 10 methylated bases or ribose 2’‐OH groups.
• The 23S rRNA has 10 pseudouridines, 1 dihydrouridine, and 12 methylated nt.