RNA Metabolism

What are the structures and functions of cellular RNAs, including noncoding RNA?

• Prokaryotic and eukaryotic cells contain abundant protein-synthesizing RNAs (messenger RNA, ribosomal RNA, and transfer RNA), and eukaryotic cells also contain abundant noncoding RNA (ncRNA).

• RNA structures are dynamic in three ways: (1) the life cycle of mRNA includes synthesis, a template for protein synthesis, and degradation; (2) RNA structure is dynamic in response to ligand binding, which shifts the equilibrium from the inactive conformation to the active conformation; and (3) the catalytic function of ribozymes is a dynamic process in that a single ribozyme molecule can catalyze the cleavage of many RNA molecules.

• The three classes of eukaryotic ncRNA are short (miRNA, siRNA, piRNA), small (snRNA, snoRNA), and long (RNaseP, TERC, lncRNA).

• Eukaryotic mRNA is transcribed by RNA polymerase II, tRNA by RNA polymerase III, and rRNA by RNA polymerases I and III.

• Transcription and translation in prokaryotes are coupled processes, whereas these two processes in eukaryotes take place in the nucleus and cytoplasm, respectively.

• Precursor mRNA in eukaryotes is transcribed and processed in the nucleus, with 5′ capping, splicing, and polyadenylation being coordinated by functions associated with RNA polymerase II.

• The percentage of the genome encoding protein-coding genes decreases in more complex organisms relative to an increase in the portion of the genome encoding ncRNA.

• Four modes of action have been attributed to lncRNA: (1) base pairing between nucleotides in the lncRNA and target RNA, (2) base pairing between lncRNA and single-stranded regions of DNA, (3) formation of functional ribonucleoprotein complexes similar to ribosomes and spliceosomes, and (4) ligand-induced conformational switches that function in signaling pathways and gene expression.

How do prokaryotic and eukaryotic RNA polymerases transcribe DNA into RNA?

• The bacterial RNA polymerase enzyme is composed of an α₂ dimer and β, β′, ω, and σ subunits. One of several transcription factors associates with RNA polymerase and directs binding of RNA polymerase to specific promoter regions.

• Bacterial promoter regions contain two conserved regions, called the −35 box and −10 box, which are responsible for σ-factor binding.

• DNase I footprinting is an experimental technique that can be used to identify regions of DNA bound by proteins, such as transcription factors or RNA polymerase.

• The eukaryotic TATA binding protein (TBP) transcription factor is responsible for recruiting all three RNA polymerases to promoter regions.

• RNA polymerase synthesizes a complement of the template strand of DNA by using ATP, CTP, UTP, and GTP. The transcription bubble contains the enzyme, a locally unwound region of DNA, and an RNA:DNA duplex of usually 8 bp.

• Bacterial transcriptional termination occurs as either a Rho-dependent or Rho-independent process.

• In RNA polymerase II transcription, the TFIID–TBP complex binds to the core promoter sequence, followed by the binding of TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH and the RNA polymerase II enzyme.

• The CTD in eukaryotic RNA polymerase II is required for coordinating precursor mRNA processing, and its functions are regulated by phosphorylation and dephosphorylation on multiple repeats of the heptapeptide sequence YSPTSPS.

• RNA polymerase II transcriptional termination is coupled to processing of the 3′ end, which involves polyadenylation by the enzyme poly(A) polymerase.

What are the mechanisms by which ribozymes generate mature rRNA and tRNA?

• Ribozymes are enzymes that contain a catalytically active RNA component. Some ribozymes are composed only of RNA, whereas others contain both RNA and protein subunits.

• The hammerhead ribozyme is a catalytic RNA that can self-cleave as well as catalyze cleavage of other RNA molecules.

• Genes encoding rRNA are transcribed as a single unit that is then cleaved and processed; in prokaryotes, rRNA genes often flank a tRNA gene.

• RNaseP is a ribonuclease present in bacteria, archaea, and eukaryotes that generates the mature 5′ end of tRNAs and cleaves some precursor rRNA transcripts.

• Precursor tRNA transcripts undergo cleavage at the 5′ and 3′ ends, addition of a CCA trinucleotide sequence to the 3′ acceptor stem, and base modification. Many tRNAs also are spliced to remove an intron in the anticodon loop.

• Processing of precursor rRNA occurs co-transcriptionally in the nucleolus, along with assembly of ribosomal proteins on the nascent transcript. Base modification of eukaryotic rRNA is catalyzed by small nucleolar RNAs (snoRNAs).

How is eukaryotic precursor mRNA spliced to generate mature mRNA?

• Eukaryotic RNA splicing occurs when an intron is removed in a transesterification reaction, which is coupled to rejoining of the 5′ and 3′ ends to generate a processed transcript.

• Group I and group II self-splicing intron reactions do not require proteins, whereas spliceosome-mediated precursor mRNA splicing involves small nuclear ribonucleoproteins (snRNPs).

• Group II intron self-splicing and spliceosome-mediated splicing give rise to an excised lariat intron structure that is degraded.

• Eukaryotic precursor mRNA introns are flanked by short conserved sequences at the 5′ and 3′ splice sites. The branch site is located 15–45 nucleotides upstream of the 3′ splice site, and in higher eukaryotes it contains a polypyrimidine tract.

• A 7-methylguanylate cap (m⁷G cap) is added to the 5′ end of RNA polymerase II transcripts and protects the mRNA from degradation by 5′ to 3′ exonucleases. The m⁷G cap also serves as a binding site for factors that direct splicing, nuclear export, and efficient translation.

• Transport of eukaryotic RNA from the nucleus to the cytoplasm requires associations with transport proteins, one of which is the protein Ras-related nuclear protein (Ran); export of mRNA from the nucleus is Ran independent.

• Targeted RNA decay is a normal cellular process that promotes mRNA turnover as a method of gene regulation. Decapping and deadenylation of the transcript are components of most mRNA decay mechanisms.

• Alternative splicing of mRNA can increase genomic complexity but can also cause disease if defective. DNA mutations can lead to a gain or loss of splice sites, resulting in alternative splicing and production of aberrant proteins.

How does RNA interference silence gene expression?

• RNA interference (RNAi) refers to gene silencing, a process that is mediated by long or short double-stranded RNA molecules that can form base pairs with a target RNA and direct its degradation or inhibit translation.

• Double-stranded RNA is cleaved into short double-stranded fragments called siRNA (21–25 nt) by the RNase III–like enzyme Dicer and loaded onto the RNA-induced silencing complex (RISC). The RISC binds to and catalyzes cleavage of a complementary target RNA, which could be mRNA or viral RNA.

• Micro RNAs (miRNAs) are short, untranslated RNAs that bind to mRNA and negatively regulate gene expression. They are encoded in the genome, transcribed by RNA polymerase II or III, and cleaved into siRNA by the Dicer enzyme.

Everyday example: CO-24: RNA Splicing Defects and Blindness