RNA Processing

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5-prime end

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3-prime end

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Poly-A tail/adenine

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Introns

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Messenger RNA (mRNA)

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RNA Processing

Difference in RNA Processing in Prokaryotes and Eukaryotes

The RNA formed after transcription is called primary RNA. In both prokaryotes and eukaryotes, primary ribosomal RNA (rRNA) and transfer RNA (tRNA) is processed.

However the primary messenger RNA (mRNA) is processed only in eukaryotes. In prokaryotes, the primary mRNA is used without any further processing. Translation, or protein synthesize, can start even before transcription is completed, as DNA is found in the cytoplasm and not encased in a nucleus. A single prokaryotic mRNA can also contain transcripts for many proteins. In eukaryotes, each mRNA can code for only one protein.

Processing

In eukaryotes, the processing of tRNA and rRNA are different from that of the mRNA, and each has its own processes. The processing of RNA that is usually described pertains to mRNA.

dna transcriptionThe mRNA is processed by capping and polyadenylation, so that the ends are not degenerated by exogenase enzymes, and to move RNA out of the nucleus into the cytoplasm. The primary mRNA also has to be spliced to produce the required sequence of bases to code for protein production.

Processing can happen during transcription (co-transcriptional) or later (post-transcriptional). 

Capping the 5' end

Capping occurs co-transcriptional at the 5' end of the RNA that is the first to be formed during transcription. After the elongation has added twenty to thirty nucleotides, a modified guanine nucleotide, the 7-methyl guanosine, is added in a unique 5' to 5' triphosphate. Two enzymes are involved in the process. One forms the unique linkage between the RNA and a guanine nucleotide, and the other adds the methyl group to guanine to give guanosine. As the exonuclease do not recognise them, they do not destroy them. This protects the 5' end of the mRNA, and helps to dock it to ribosomes once it is out in the cytoplasm.

Polyadenylation of the 3' end


The RNA synthesis is stopped when the sequence AAUAAA is encountered. Four multi-protein complexes are involved in recognizing the AAUAAA sequence, while the actual cleavage of the  RNA downstream to the sequence is done by a ribonuclease. Some of the multi-protein complexes also stimulate polyadenylation, which is carried out by the enzyme polymerase A. Polymerase A adds a poly-A chain made of 100 to 200 adenine (A) nucleotides to the 3' end. The poly-A tail can be degraded by exonuclease, but this happens slowly and the mRNA can still fulfill its role in protein production.

This poly-A tail makes RNA more stable and guides it out of the nucleus through nuclear pores to the cytoplasm. Polyadenylation is partly post-transcriptional. 

Splicing

DNA in eukaryotes have short exons that code for a gene, separated by long non-coding sections called introns. All the three types of eukaryotic RNA have exons and introns, and these introns have to be removed to make RNA functional. The process by which they are removed is called splicing, and is post-transcriptional.

Each intron is recognized by a GU sequence at the 5' end (called 5' splice site), and the AG sequence at the 3' end (or 3' splice site). Splicing is done by spliceosomes, which are a complex of 500 proteins and 5 nuclear snRNA. These are called U1, U2, U3, U4 and U5. The U1 binds to the 5' splice site, and U2 to the branch site A near the 3' splice site. Then U3, U4 and U5 bind to the rest of the intron, bringing the two ends of the intron together in a loop. The 5' end is spliced and attached to the branch site. Then the 3' end is cut, and the looped intron is removed by the spliceosome complex, bringing exon ends together. The exon ends bind through covalent bonds.

In a process called alternative splicing, not all exons in a primary mRNA are bound. Different exons can be left out, so that the combination of exons included provides a different sequence, and therefore code for different protein. So depending on how exons are spliced or included, a preliminary RNA strand can yield many different protein isoforms with varying biological functions. About 60% of human RNA go through alternative splicing. As a single gene is used to produce many proteins without changes in the genome, it is also an evolutionary advantage. The number of exons and the length of RNA can vary. For example, the dystrophin gene is 2500 kb large, and the mRNA 14 kb, and has 79 exons.

In prokaryotic mRNA, the introns are self-splicing and do not need extra processing.

conserved_polyadenylation_sequence Summary

Processing of ribosomal RNA (rRNA) and transfer RNA (tRNA) occurs in both prokaryotes and eukaryotes. Only mRNA in eukaryotes undergo processing that involves three steps: capping, polyadenylation, and splicing. Capping adds a 7-methyl guanosine to the 5' end, and polyadenylation adds a poly-A tail to the 3' end of the primary RNA. These processes protect the two ends and help move the RNA out of the nucleus to ribosomes in the cytoplasm. Splicing removes non-coding introns and joins exons to give a functional coding sequence. Alternative splicing determines which exons are included, so that various protein isoforms can be produced from a single gene.

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