Re: i was wondering how a premature stop codon could cause mRNA instability

Aloha Leilani,

Unfortunately, the answer to your question is necessarily a long one due to the complicated nature of mRNA regulation and utilization, and the fact that there are several outcomes which are equally possible. So please bear with me.

In order to answer your question regarding how a premature stop codon may result in mRNA instability, we have to first understand the life cycle of messenger RNA and secondly how a premature stop codon may alter that normal life cycle. In part the differences may be due to the time scale, over which bacteria and higher organisms respond to changes in the environment, and what that means in the context of the complexity of an organism.

Bacteria, which can replicate as often as once an hour, must be able respond to changes in the environment (temperature, nutrition, antibiotic attacks, etc.) extremely quickly, so rapid regulation of which proteins are made and when they are made, is critical to bacterial survival. Protein synthesis is tightly controlled through continuous degradation of mRNA; protein synthesis is only initiated when a signal is sent to turn on mRNA synthesis, and is quickly shut down by constant RNase degradation. On the other end of the spectrum, higher organisms have evolved a complicated cellular structure and higher-ordered structures (tissues, organs, etc.); the biological response to changes in the environment are comparatively much slower than that of bacteria. For example, the enzymes required to metabolize certain things we eat may not be synthesized in the liver until hours after we ingest food and may be required for long periods of time after that. While protein synthesis is also controlled in part through mRNA degradation, it is a much slower process than in bacteria.

Though the overall process of mRNA synthesis and degradation is similar in prokaryotes and eukaryotes, there are major differences between what happens to mRNA in bacteria and in eukaryotic cells, and this is related to compartmentalization. In bacteria, which have no nuclei, mRNA is both transcribed and translated in the same cellular compartment; the two processes occur virtually simultaneously. Ribosomes attach to bacterial mRNA even before transcription has been completed. Protein synthesis is turned off by proteins called RNases, which function to rapidly degrade mRNA. If you look in any advanced molecular biology textbook, I'm sure you will find a picture of mRNA being synthesized by an RNA polymerase, with ribosomes already attaching themselves to the newly synthesized piece of mRNA and perhaps degradation already underway at the 5' end via RNase digestion. From start to finish, the whole process of transcription, translation, and mRNA degradation is typically finished in two minutes. So a signal comes into the cell which is interpreted as a need for a particular protein, turning on mRNA synthesis, a few proteins get made from each mRNA molecule by several ribosomes, then the mRNA is degraded. If more protein is needed, more mRNA must be made. While this may seem energetically expensive, it is an efficient use of resources compared to the amount of energy that would be used up making unnecessary proteins.

This cost-effective strategy is also present in eukaryotes. However, the timing of events is extended due to the fact that synthesis and processing of mRNA occurs in the nucleus while ribosomal protein synthesis occurs in the cytoplasm. In the nucleus, the nascent pre-mRNA transcript is spliced (intron removal), 5' end-capped, and polyadenylated in the 3' untranslated region. These modifications occur in 100% of mRNA molecules that are exported out of the nucleus into the cytoplasm, suggesting that nuclear degradation occurs if all of these processing events are not completed. Once end-capped, spliced, and polyadenylated, eukaryotic mRNA is very stable to premature degradation, and is exported to the cytoplasm through the nuclear pore complexes. (The textbook I looked in cited a 20 minute delay between initial transcription and export of mature mRNA). Once out of the nucleus, a ribosome recognizes and associates with a mature mRNA, translating the message into proteins. The same transcription regulatory mechanism of mRNA degradation at the hands of RNases operates in eukaryotes, but the process is much slower. On average, cytoplasmic mRNAs are stable for many hours.

Back to your question, the presence of a premature stop codon in eukaryotic mRNA can give rise to a number of outcomes, including targeted mRNA degradation. Eukaryotic cells have evolved conserved proofreading mechanisms to get rid of incomplete and potentially deleterious proteins. For example, the nonsense-mediated mRNA decay (NMD) pathway (characterized in yeast, a eukaryote commonly used in genetic studies) is an example of a surveillance mechanism that monitors premature translation termination and promotes degradation of aberrant transcripts that code for nonfunctional or even harmful proteins. There are several models for how this pathway determines if a stop codon is correct or premature, all of which involve the function of proteins that normally bind to mRNA. For example, the exon-exon junction is 'tagged' by a particular protein in the nucleus that lets the splicing machinery know a splicing event has occurred already (this prevents loss of exons through additional splicing events); this protein is believed to remain associated with a given mRNA up until the point that a translating ribosome knocks it off. One model of NMD suggests a stop codon that comes before a 'tagged' exon-exon junction is a signal for mRNA degradation.

On the other hand, translation stop codon read-through is another common outcome of a premature stop codon. Normal translational termination requires a protein associated with the stop codon and a ribosomal pause site which combine to dissociate the ribosome, thereby halting protein synthesis. A premature stop codon may lack the necessary pause site, allowing the ribosome to pass through it without terminating translation; read-through is achieved by codon skipping, frame-shifting, or utilization of suppressor tRNAs. The misreading of termination codons is achieved by a variety of naturally occurring suppressor tRNAs that allow the ribosome to insert an amino acid in the growing chain, though termination is called for based on the mRNA sequence. All of the nonsense suppressors characterized to date are normal cellular tRNAs that are primarily needed for reading their cognate sense codons. The end result is the ribosome may be able to avoid the premature stop codon, producing functional proteins.

Before I go on, I wanted to mention that there are some REALLY recent studies (so recent that you won't read about it in textbooks) that suggest that fully processed mRNA is 'proof-read' in the nucleus. It has been proposed that nuclear ribosomes make a small amount of test protein to make certain that it encodes for a functional protein either just before or just after it is exported from the nucleus. However, this is a VERY recent and controversial result, is still an area of new research and is by no means guaranteed to be correct. It would suggest that if a mutation results in a premature stop codon in the open reading from of a mRNA, or even if an intron is not spliced out properly, a protein fragment may be synthesized which is either non-functional or unfolded, and may signal that a particular mRNA should be degraded. As I said, it's controversial and is not typically discussed as the primary reason mRNAs encoding non-functional proteins may be actively targeted for degradation.

In summary, a premature stop codon in prokaryotes does very little to affect the naturally short lifetime of mRNA in bacteria. It is highly probable that the protein it codes for may not be functional, but the mRNA is normally degraded quickly anyway. A premature stop codon in eukaryotes will in general increase the likelihood that a given mRNA will be targeted for early degradation via the normal mRNA clearance pathways. However, it is also likely that the premature stop codon in eukaryotes may be avoided through codon skipping, frame-shift mutations, or through the utilization of suppressor tRNAs.

Thanks for your interesting question.

Regards,
Dr. James Kranz