What are the key structures and functions of eukaryotic 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).
● Three classes of eukaryotic ncRNA have been characterized: 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.
● Precursor mRNA in eukaryotes is transcribed and processed in the nucleus, with 5’ capping, splicing, and 3’ 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 riboswitches that function in signaling pathways and gene expression.
What are the molecular determinants of RNA synthesis?
● The bacterial RNA polymerase holoenzyme is composed of an α2 dimer and β, β9, and ω subunits. One of several σ transcription factors associates with the holoenzyme and directs binding of RNA polymerase to specific promoter regions.
● Bacterial promoter regions contain two conserved regions, called the 235 box and 210 box, which are responsible for σ-factor binding.
● 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 using ATP, CTP, UTP, and GTP. The transcription bubble contains the enzyme, a locally unwound region of DNA, and an RNA:DNA hybrid helix of usually 8 bp.
● 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.
What are the molecular determinants of eukaryotic RNA processing?
● 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.
● Alternative splicing of mRNA can increase genomic complexity but can also cause disease. DNA mutations can lead to a gain or loss of splice sites, resulting in alternative splicing and production of aberrant proteins.
What is the biochemistry of RNA-mediated gene silencing by siRNA and miRNA?
● 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 bp) 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.