Eukaryotic Pre-mRNA Processing

Processing of Eukaryotic Pre-mRNA

• Pre-mRNPs must be polyadenylated before they are called nuclear mRNPs.
• The RNA recognition motif, found in hnRNP proteins, is also known as the RNA-binding domain.
• Expressing hnRNP C protein (tagged with green fluorescent protein) and hnRNP A1 protein (tagged with red fluorescent protein) in Xenopus cells results in green and red fluorescence in the nucleus.
• Mutations affecting SR protein binding to exonic splicing enhancers can cause exon skipping, leading to nonfunctional proteins
• SR proteins contain an RS domain involved in protein binding.
• Sequencing small RNAs from metazoan cells reveals low levels of short, capped RNAs transcribed from both DNA strands, described as pervasive transcription.
• AAUAAA is the consensus sequence for poly(A) addition.
• Histone mRNAs lack poly(A) tails, introns, and a 3' UTR.
• Splicing involves two transesterification reactions.
• Splice sites in pre-mRNA have two universally conserved sequences at the ends of introns.
• Splicing joins two exon sequences.
• The branch-point A residue in lariat formation is part of the intron.
• The order of steps for mature mRNA production is: initiation of transcription, addition of 5' cap, splicing, addition of poly(A) tail, and transport to the cytoplasm.
• Spliceosome components include pre-mRNA, proteins reacting with sera from systemic lupus erythematosus patients, and U1 snRNA interacting with the 5' splice site

Regulation of Pre-mRNA Processing

• snRNA (small nuclear RNA) functions in removing introns from pre-RNAs.
• Alternative splicing explains the appearance of multiple mRNAs from a gene encoding a Ca2+-activated K+ channel in bird auditory hair cells.
• Sex-lethal protein in Drosophila is a splicing regulatory factor.

Transport of mRNA Across the Nuclear Envelope

• Excision of group II introns does not require enzymes.
• RNA editing is a post-transcriptional alteration of mRNA sequences.
• Export of mRNAs outside the nucleus necessitates post-translational modification by phosphorylation of several proteins.
• Retroviruses have a constitutive transport element allowing unspliced RNAs to be exported into the cytoplasm.
• RNA editing in protozoans does not occur within the nucleus
• hnRNA participates in nuclear export of mRNA.
• Transport of unspliced HIV mRNA from the nucleus to the cytoplasm of host cells is promoted by the viral protein Rev.

Cytoplasmic Mechanisms of Post-transcriptional Control

• The RISC complex contains the protein Argonaute.
• Knocking out the dicer gene in mammals would lead to a loss of miRNAs.
• An siRNA-containing RISC complex is distinguished from an miRNA-containing RISC complex because the siRNA base-pairs perfectly with its target mRNA.
• The mammalian target of rapamycin (mTOR) complex 1 is not directly involved in protein degradation.
• The autophagosome plays a key role in the digestion of ribosomes, mitochondria, and other organelles.
• Polysomes do not form at p-bodies.
• Nonsense-mediated decay, non-stop decay and no-go decay are all considered mRNA surveillance mechanisms.
• MicroRNAs play a key role in translation repression.
• Capping does not take part in the degradation process of eukaryotic mRNAs

Processing of rRNA and tRNA

• P-bodies are not found in the nucleus
• Synthesis of pre-rRNA occurs in the nucleolus.
• The 45S pre-rRNA molecule can organize a nucleolus when present in a single copy, is encoded by tandemly arranged genes, and is methylated on specific bases.
• A ribozyme is an RNA sequence that uses Mg2+ ions as a cofactor, has catalytic ability to cleave RNA, and acts in the spliceosome.

Processing of Eukaryotic Pre-mRNA - Testing on Concepts

• A capping enzyme removes the γ-phosphate from the 5' end of nascent RNA emerging from RNA polymerase II.
• A separate subunit of the capping enzyme transfers a GMP moiety from a GTP donor to the 5' diphosphate of the nascent transcript, forming a 5'-5' triphosphate structure.
• Separate enzymes then methylate the guanine's N7 position and the riboses' 2' oxygen.
• Poly(A) signal (often AAUAAA) binds CPSF.
• Poly(A) site is where cleavage occurs and A residues are added.
• The G/U-rich region binds cleavage stimulatory factor (CStF).
• CPSF binding initiates pre-mRNA polyadenylation.
• CStF binds to the CPSF-RNA complex, stabilized by a GU-rich sequence interaction.
• Poly(A) polymerase binds the complex, stimulating RNA cleavage and A residue addition.
• PABII enhances A residue polymerization.
• Poly(A) tail length is restricted to about 200 nucleotides via an unknown mechanism.
• hnRNP proteins are major components of heterogeneous nuclear RNA particles, including unspliced nuclear mRNA and other nuclear RNAs
• hnRNP proteins are identified by UV irradiation, which causes covalent cross-links between RNA and associated proteins.
• Chromatography of nuclear extracts from irradiated cells on an oligo-dT cellulose column recovers cross-linked proteins.
• snRNP-dependent splicing likely evolved from self-splicing introns.
• snRNP-mediated splicing allows regulation and alternative RNA splicing, unlike self-splicing.
• U1 snRNP interacts with pre-mRNA for splicing due to complementarity between the 5' end of introns and a sequence near the 5' end of U1 snRNA.
• Specific antiserum prevents in vitro splicing.
• Synthetic oligonucleotides compete with normal U1 snRNA, preventing splicing.
• Mutations in either the 5' splice site or U1 snRNA prevent splicing, but compensatory mutations restore splicing.
• Anti-U2 snRNP immunoprecipitates four intermediates in pre-mRNA splicing: a structure joining exons with the intron, a structure with the excised intron in lariat form, pre-mRNA with U2 snRNP bound to the 5' end of the intron, and a complex of pre-mRNA, U1 snRNP, and U2 snRNP bound to the branch site.
• Anti-U2AF immunoprecipitates the same complexes as it assists U2 snRNP in binding.
• U2 snRNA base-pairs to a short sequence near the branch-point A in yeast introns, while U2AF promotes U2 snRNA binding in higher eukaryotes with less conserved sequences.
• Heterozygous U2AF knockout mice are likely viable due to sufficient U2AF production, while homozygous U2AF knockout mutation would be lethal due to the necessity for proper splicing.

Regulation of Pre-mRNA Processing - Testing on Concepts

• Early in development, Drosophila females express sxl mRNA from the Pe promoter, producing early Sxl protein after splicing of exons 1 and 2.
• Subsequently, the Pl promoter drives expression of sxl mRNA with exons 1-4, and Sxl protein prevents splicing of exons 2 and 3.
• The resulting mRNA containing exons 1, 2, and 4 is translated into functional late Sxl protein, which continues to bind the sxl pre-mRNA.

Transport of mRNA Across the Nuclear Envelope - Testing on Concepts

• HIV bypasses normal restrictions mRNA transport via the protein Rev.
• Rev protein binds to the Rev response element (RRE) and is exported from the host cell nucleus.

Cytoplasmic Mechanisms of Post-Transcriptional Control - Testing on Concepts

• Oocytes contain stored mRNAs required for early embryonic development that remain untranslated until fertilization.
• Stored mRNAs in oocytes have short poly(A) tails (~20-40 residues), which bind only a few PABPI molecules i.e. not enough to interact with eIF4G.
• After fertilization, the poly(A) tail increases in length with an additional ≈150 A residues.
• PABPI binds to eIF4G in a multimeric complex with eIF4E and other factors at the 5' cap, forming a stable conformation.

Processing of rRNA and tRNA - Testing on Concepts

• Researchers insert binding sites for RNA binding proteins into manipulate the RNA sequence, then fuse the RNA binding proteins with fluorescent proteins.
• Using these probes the subcellular locations of RNA molecules can then be determined .
• Tissue-specific expression of antisense RNA can selectively shut down protein production by coupling it to a promoter specific to anthers, which are parts of the flower that produce pollen.
• Catalytic RNA (ribozyme) can prevent protein expression via some RNAs that have both sequence-specific base pairing and RNase activity.
• A 400-nucleotide-long intron from Tetrahymena rRNA (a group I self-splicing RNA) folds and binds a guanine nucleotide and a substrate RNA chain and catalyzes the covalent attachment of the G to cleave the substrate RNA.
• A properly designed catalytic RNA can bind to any substrate RNA and sever it at a specific site.
• A DNA sequence encoding a catalytic RNA engineered results in the tissue-specific synthesis of a ribozyme destroying a differentiation-specific mRNA required for pollen production.
• Pre-tRNA splicing differs as it does not involve spliceosomes. An endonuclease-catalyzed reaction excises the intron and a 2',3'-cyclic monophosphate ester forms on the cleaved end of the 5' exon. A hydrolysis of GTP and ATP joins the two exons. In contrast, pre-mRNA splicing does not require GTP and releases the intron as a lariat structure.