Post-Transcriptional Regulation

Post-Transcriptional Control of Gene Expression

Recall the Central Dogma by which instructions in the form of DNA in the cell are made into RNA before eventually being made into proteins (Fig. 1).

During transcription, DNA is turned into heterogeneous nuclear RNA (hnRNA). Following transcription, a series of enzyme-catalyzed modifications called post-transcriptional modifications occur to convert hnRNA into functional messenger RNA (mRNA).

In eukaryotic cells, both transcription and post-transcriptional modifications occur in the nucleus while translation occurs in the cytoplasm. As viruses and bacteria use single-stranded RNA to replicate inside the cytoplasm, eukaryotic cells have developed mechanisms to degrade single-stranded RNA as a protective mechanism.

If hnRNAs without post-transcriptional modifications are released into the cytoplasm, they would be degraded by the cell's own degradation machinery. Post-transcriptional modifications in the nucleus allow mRNA to avoid self-degradation and survive in the harsh environment of the cytoplasm.

In contrast, prokaryotic cells do not have organelles or a nucleus. Therefore, both transcription and translation occur in the cytoplasm. To prevent RNA from being degraded in the cytoplasm, prokaryotes begin translation before transcription has been completed.

Therefore, prokaryotic cells do not have post-transcriptional modifications, instead, prokaryotes have alternative mechanisms to validate mRNA quality and prevent viral replication.

Types of Post-Transcriptional Regulation

There are three post-transcriptional modifications that must occur to the hnRNA before it can be released into the cytoplasm as a mature mRNA:

1. Addition of a 5' GTP cap.
2. Addition of a 3' poly-A tail.
3. Removal of introns and retention of exons.

The details of each modification will be discussed in the next section.

Post-Transcriptional Regulation in Eukaryotes

Addition of a 5' GTP cap

Recall that RNA and DNA have directionality with a 5' end and a 3' end. At the end of the 5' end of hnRNA, a 7-methylguanylate-triphosphate cap also known as a 5' GTP cap is added. The 5' GTP cap is a modified guanine nucleotide with an additional methyl group at the 7th position of guanine.

The addition of the 5' GTP cap is catalyzed by three enzymes which are part of the CAP-binding complex. The CAP-binding complex will connect the 5' carbon of the GTP cap to the 5' carbon of the first nucleotide to create a 5'-to-5' triphosphate bridge.

There are three overall functions of the 5' GTP cap:

1. To aid in RNA processing and export of the RNA from the nucleus.

2. To act as a marker to orient the mRNA during translation in the cytoplasm.

3. To protect the mRNA from degradation.

Addition of a 3' poly-A tail

The polyadenosyl (poly-A) tail is added to the 3' end of hnRNA to protect the RNA transcript from rapid degradation in the cytoplasm.

Once any single-stranded RNA strand enters the cytoplasm, it will be rapidly degraded by the cell's degradation machinery known as 3'-exonucleases. The 3' exonucleases rapidly degrade single-stranded RNA beginning from its 3' end as a protective mechanism against viral and bacterial RNA transcripts. Once the mRNA is released into the cytoplasm, it too will be degraded by 3'-exonucleases.

However, the poly-A tail acts as a buffer that will be degraded before the coding information on the mRNA. The longer the poly-A tail, the longer that the mRNA transcript can survive in the cytoplasm. The poly-A tail is also critical for the export of mature mRNA from the nucleus into the cytoplasm.

The addition of the 5' GTP cap and the 3' poly-A tail is illustrated in Figure 2 below.

Removal of Introns

Recall that eukaryotic genes are made of coding sequences known as exons and non-coding sequences known as introns. Often introns are spaced between exons, therefore, following the transcription of DNA into RNA, the introns must be removed and the exons must be reassembled into one mRNA strand (Fig. 3).

The process to remove introns is known as splicing. Splicing is performed by a complex called the spliceosome which itself is made up of non-coding RNA called small nuclear RNA (snRNA) and proteins called small nuclear ribonucleoprotein (snRNPs).

Together, they make up the spliceosome which will recognize the boundaries of the intron and excise them from the RNA to be degraded. The final product is an RNA transcript that is made up of only exons.

Some eukaryotic genes, however, are made up of unique combinations of exons. This means that not all exons are transcribed into a mature mRNA.

In Figure 4, different exons are included in the final RNA transcript. On the left, exon #2 but not exon #3 may be included in mRNA #1 while the opposite may be true for mRNA #2. This selective inclusion of exons is known as alternative splicing. Alternative splicing allows for different forms of the protein to be made from the same DNA sequence.

Post-Transcriptional Regulation Examples

Recall that all cells in the human body have the same genome, however, not all the cells look the same nor do they express the same proteins.

One main reason for this difference is that different cells express different genes. Another reason is alternative splicing. Cells can splice RNA transcripts differently to make different proteins from the same gene. In fact, 75% of human genes produce multiple forms of the same proteins which ultimately increases the coding potential of the genome.

Post-transcriptional modifications are also critical for sex determination in the fruit fly. Just like humans, there are certain genes in fruit flies that can be alternatively spliced to form different versions of the same protein: one gene in particular called the doublesex gene is responsible for determining the sex of a fruit fly. Following alternative splicing, one form of the doublesex protein will inhibit genes involved in male fruit fly development.

However, another form of the protein will inhibit female fruit fly development. Importantly, this example illustrates how post-transcriptional modifications can have drastic effects on the organism even though the gene itself can be identical.

Ultimately, alternative splicing increases the coding potential of the genome. Rather than having two independent gene pathways, the same gene can be expressed differently through alternative splicing to have opposite effects.

Post-Transcriptional Regulation in Prokaryote

Unlike eukaryotes, prokaryotes do not have post-transcriptional modifications. The 5' GTP cap, 3' poly-A tail, and introns are unique to eukaryotes. One reason may be due to the spatial separation between RNA and proteins in eukaryotes.

Unlike eukaryotes, prokaryotes do not have a nucleus so both transcription and translation occur in the cytoplasm. This means that RNA does not need to be transported from the nucleus into the cytoplasm. Without this need for transportation, translation can occur on the mRNA transcript simultaneously as the mRNA transcript is being built.

Therefore, mRNA would not need to be as carefully guarded against degradation since translation starts immediately. Without the need for protection and transport, post-transcriptional modifications would not be needed.

However, prokaryotes have alternative ways to check the quality of the mRNA. If an incomplete or broken mRNA is being translated by the ribosome, an 11 amino acid tag will be added to the end of the newly synthesized polypeptide. This 11 amino tag acts as a signal to proteases within the prokaryotic cell to destroy the entire protein. By destroying the protein, it prevents incomplete or broken proteins from lingering in the cell.

Additionally, without post-transcription modifications, mRNAs are rapidly degraded in the prokaryotic cell; therefore, translation must occur while a new mRNA transcript is being synthesized.