M2C: Rna Processing

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Exons

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Coding regions of genes. On average a human gene consists of ~8 exons, each about 145 bases. Introns (non-coding regions) are interspersed between exons. These are often 10 times or more bigger than exons.

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RNA Processing

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RNA polymerase II is unable to distinguish between introns and exons. Processing begins as soon as nascent transcript emerges from polymerase. Introns must be "spliced out" before translation can occur. 5' and 3' (polyA addition) end capping must also occur. RNA processing is necessary for mRNA stability, transport, splicing, and/or translation. Each step is a potential site for regulation of gene expression.

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Final modifications of mature mRNA

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Mature mRNA has a 5'-7-methyl-guanine cap, a 5' untranslated region, a continuous coding region, a 3' untranslated region, and a polyA tail.

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Eukaryotic Promoters

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Many eukaryotic genes have alternative promoters. Variation is decided by type of cell or tissue, or under different metabolic conditions.

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Beginning of RNAPII transcript processing

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Occurs as soon as nascent transcript emerges from polymerase. The phosphorylated C-terminal domain (CTD) of RNAP II is recognized during many of the events in RNA processing, probably because RNAPII needs CTD to transcribe at authentic promoters. When the transcript is ~25 nucleotides long, CTD binds to enzymes that modify the 5' end and covalently add guanosine with an unusual 5'-5' linkage. This guanosine and nearby ribose are then methylated to form the 5' 7-methyl-guanine cap.

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Purpose of the 5' 7-methyl-guanine cap

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1. Marks the RNA molecule as an mRNA precursor.
2. Protects the RNA from 5'-3' exoribonucleases in the cell.
3. Serves as a binding site for Cap Binding Protein (CBP).

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Heterogeneous Ribonuclear Proteins (hnRNPs)

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Several hundred exist and perform various functions in RNA transcript processing. Some bind to RNA transiently while others stay with it into the nucleus and/or into the cytoplasm. Most have at least two domains, one for attachment to the RNA and another to interact with other proteins (like splicing factors). Some also have additional catalytic sites.

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RNA Splicing

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Introns must be removed to produce a continuous and open reading frame for protein coding.

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Splice site recognition

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Splice sites are recognized by comparing a mature mRNA with that of the gene encoding the RNA. Certain consensus sequences are conserved at or near the splice site junctions. Mutations to this splice site will abolish splicing there unless there is a cryptic branch point near the authentic site.

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Cryptic Branch Sites

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Work very similarly to authentic splice sites. If present, are found near 3' splice site. The product of splicing is normal if a cryptic splice site is used. This tells us that the point of a branch site is to find the nearest 3' splice site in conjunction to the 5' site.

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The Chemical Reaction of Splicing

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Consists of two sequential transesterification reactions between components of the sugar-phosphate backbone of the RNA. First occurs when the 2'OH of the branch site A residue attacks the phosphodiester bond linking the final G residue in exon 1 with the first G in the intron. The result is an unusual 5'-2' phosphodiester bond linking the 5' of the intron to the branch point A. Exon 1 is no longer covalently linked to the to the rest of the pre-mRNA, but is held in place by splicosome components. The second transesterification reaction: the 3'-OH of exon 1 attacks the phosphodiesterase bond linking the exon with the 5' end of exon 2. This frees the intron as a lariat structure. The exons are now correctly linked.

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What happens to introns once they are spliced out of pre-mRNA

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Release as lariat structures. Are degraded or may contribute to microRNA formation.

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The Spliceosome

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Five RNA-protein complexes called snRNPs. U1-snRNP, U2-snRNP, U4-snRNP, U5-snRNP, U6-snRNP. It is the snRNA component of snRNPs that have the sequence and structure recognition of the intron-exon junction and branch point. Spliceosome have over 100 protein components and are larger (heavier) than ribosomes.

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Lupus Erythematosus

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Seven proteins in snRNPs that form a core that is recognised by Sm antibodies in pts with this autoimmune disease.

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Function of snRNPs

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U1-snRNP base-pairs with the 5' splice site. Then, U2AF attaches to polypyrimidine sequence at the 3'splice site. Only when this happens will U2-snRNP bind stably to the branch point. Next a trimer including U4, U5, and U6 snRNPs bind. Both transesterification reactions are catalyzed by the U2/U6 complex.

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Exon Junction Complex

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After splicing, certain hnRNPs remain attached at exon-exon junction. Play role in exporting spliced RNA out of nucleus and quality control mechanism to degrade improperly spliced RNA.

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Retinitis Pigmentosa

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Heterogeneous disease effecting ~1:4000. Progressive retinal degredation and eventual total blindness due to loss of photoreceptor cells. ~10% are due to mutations is genes coding for proteins that are part of the spliceosome. High demand for opsin synthesis makes retinal cells particularly sensitive to slowed or abberant splicing.

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Spinal Muscular Atrophy

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Autosomal recessive disorder that is a common cause of infant death. Caused by deletion of SMN1 (survival of motor neurons) gene. SMN1 protein is involved in the assemebly of the splicing snRNPs and also snoRNPs. Partial deletion causes life to be possible but musculo-neural depredation over time and inhibited development.

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Exon Definition

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Although splicing interactions happen across the intron, interactions that define 5' and 3' splice site (defining exon) occur across the exon. U2 and U1 snRNPs initially bind to the 3' splice site (5' of exon). U1 then scans downstream 5' splice site within 300 nuceotides of 3' site. If one is found, U1 is bound and initiates the spliceosome complex. This defines the exon.

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Exonic Splicing Enhancers

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Bind proteins that stimulate splicing at specific (weak) splice sites. SR proteins are best defined. Interact and bind with U2AF at suboptimal 3' splice sites.

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Exonic Splicing Suppressors

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Repress splicing at specific sites. hnRNPs work at potential 3' splice sites and repress splicing there.

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Alternative Splicing of pre-RNA

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~60% of human pre-RNA can be spliced in multiple different ways due to the presence of multiple weak splice sites. Utilization of specific splice sites is driven by age, sex, environmental stimuli, metabolic state, etc. Alternative splicing expands the repertoire of the human genome. ~25,000 genes can encode over 300-500 thousand proteins. Can also cause many single bp mutations that can result in human genetic diseases.

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Transcription Termination and Polyadenylation (PolyA) of pre-mRNAs

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There is not a specific signal for transcription termination for RNAPII. Therefore, 3' ends of pre-mRNA are generated by cleavage at a specific sequence and then polyadenylated (multiple adenines added). Polyadenylation requires simultaneous recognition of two conserved sequence elements 40-50 nucleotides apart in the newly transcribed mRNA. These sequences are binding sites for multi-subunit protein complexes called CPSF (cleavage and polyadenylation specificity factor) and CstF (Cleavage stimulation Factor).

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CPSF and CstF function in polyadenylation and 3' cleavage

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Both complexes (Cleavage and polyadenylation specificity factor and Cleavage stimulation factor) associate with RNAPII throughout elongation. As polymerase passes their recognition sequences, they drop off. Interaction between CPSF and CstF bends the DNA and recruits cleavage factors and poly(A) polymerase (PAP). Only after all parts of complex are assembled (CPSF, CstF, CFs, and PAP) can the cleavage of the G/U rich site be completed. Cleavage and polyadenylation are linked events, thus protecting against exoribonucleases. Polyadenylation terminates when tail is ~200-250 residues long. During polymerization, poly(A)-binding protein II (PABPII) binds to the poly A tail. This protein is important in mRNA transport out of the nucleus.

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Importance of polyA tails

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Failure to polyadenylate prevents the appearance of mature mRNA in the cytoplasm. More than half of human genes contain multiple polyadenylation sites creating variable mRNA and protein products. This alternative polyA creation also means alternative splicing sites and uses alternative promoters. There also seems to be a specific tissue bias toward different cleavage and polyA sites.

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Export of mRNA from the nucleus

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RNA export factor is a component of the exon junction complex (EJC) that is found at each exon-exon junction in fully processed mRNA. This exon export factor as well as an SR proteins interact with the mRNP transporter, which carries the mRNP to the nuclear pore and out of the nucleus. As the mRNP leaves the nuclear pore, some of the proteins components are shed into the nucleoplasm. Others are shed once the mRNP moves into the cytoplasm. These shed proteins recycle back to the nucleus where they can be reused.

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Proteins that interact with mRNP in the cytoplasm

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eIF4E replaces the CAP binding protein during the first round of translation of the mature mRNA. PolyA binding protein I replaces PolyA binding protein II. These proteins interact during translation to increase the frequency of ribosome recruitment of the mRNA.

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Ribosomes

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Large ribonucleoprotein particles that carry out directed protein synthesis. Eukaryote ribosomes consist of four rRNAs and about 70 ribosomal proteins. All three RNA polymerases are involved.

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rRNA synthesis

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5S rRNA genes are transcribed by RNA polymerse III inside the nucleoplasm (not in the nucleolus). The other three rRNAs are transcribed by RNAP I in the nucleolus. They are transcribed as a single pre-rRNA from a group of tandem repeat genes. This initial transcript is called 45S primary transcript. It is processed using endo and exonucleolytic cleavages as well as base modifications to become mature 18S, 5.8S, and 25S rRNAs.

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pre-rRNA modification and processing

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Pre-rRNAs are bound to proteins as soon as they are synthesized, but nucleolytic step does not occur until transcription is nearly complete. Base modifications (like methylation) occur early.

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snoRNA

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"Guide RNA" identify sites for modification on pre-rRNA. A group of small nucleolar RNAs responsible for determining positions of sites of 2'-O-methylation and of pseudouridine formation, and for endonucleolytic cleavage during rRNA processing.

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Types and functions of snoRNA

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1. Box C+D snoRNA base pair with sites on pre-rRNA and direct an enzyme called methyltransferase to that site to methylate ribose.
2. H+ACA snoRNA recruit enzyme to convert uridine to pseudouridine.
3. U3snoRNA makes initial endonucleolytic incisions, and with others, carry out cleavage reactions.

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History of snoRNA

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snoRNA is newly discovered because it is processed from introns (lariate structures). While previously introns were always considered "junk", it turns out that some serve important roles. The modifications made by snoRNAs are found deep in the folded structure of rRNA and are conserved in most rRNA structures, leading us to believe that they are quite important to the function of the structure. We do not know why, however.

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Ribosomal assembly

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Ribosomal proteins begin to associate with pre-rRNA before its transcription or cleavage is complete. Ribosomal proteins are synthesized by cytplasmic ribosomes (transcribed by POLII) and transported back to the nucleolus for ribosome assembly. Once the 32S rRNA precurser is cleaved, the small ribosomal subunit is transported out of the nucleolus toward the cytoplasm. The 5.8S and 27S precurser continue through cleavage steps. Eventually the 40S subunit is exported to cytoplasm followed by the 60S subunit.

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Amount of rRNA

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Depends on physiological state of the cell. In growing mammals, 40% of RNA synthesis is rRNA. Ribosomal protein sythesis follows same trends.

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rRNA and disease (general)

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Because there are over 1000 genes coding for rRNA, a mutation to a single one is unlikely to compromise rRNA processing and function. Loss of a single ribosomal protein, however, can decrease the total number of ribosomal proteins for a subunit and therefore destabilize the entire ribosomal subunit.

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Diamond Blackfan Anemia (DBA)

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A rare autosomal dominant bone failure syndrome that effects the end stage of RBC differentiation. Is caused by any of a number of mutations of genes coding for ribosomal proteins. Can be a mutation effecting the large or small subunit. Thought that because there is much protein sythesis in differentiating erythrocytes, anemia is primary symptom.

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Processing of tRNA

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Pre-tRNAs are synthesized by RNA polymerase III in the nucleus and undergo many modifications to the 5' and 3' ends. Mature tRNAs are usually 75-80 nucleotides long and about 10% is modified.
1. U residues of the 3' end are replaced by CCA sequence which is required for the charging of aminoacyl-tRNA synthetases.
2. Methylation of specific purine bases and the 2'-OH of certain ribose groups.
3. Modification of specific uridine bases to pseudouridine, dihydrouridine, and ribothymidine bases.

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Pre-tRNA introns

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Very different from pre-mRNA introns. Don't have same kind of consensus splice sites. Both 3' and 5' incisions are made simultaneously by an endonuclease to excise intron in a single step. Hydrolysis of ATP and GTP is required to ligate the two halves of tRNA back together. No other RNA is involved in the process.

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Transport of tRNA

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After processing in the nucleoplasm, mature tRNA is transported to the cytoplasm through the nuclear pore complex.