In this mRNA processing steps in eukaryotes post we have briefly explained about mRNA processing in eukaryotes: 5′ capping, 3′ Poly-A Tail, Pre-mRNA splicing, discovery of introns, and intron processing.
mRNA processing in eukaryotes goes through a lot of mRNA processing in eukaryotes happened before being translated. Additional steps in the maturation process of mRNAs in eukaryotes produce molecules with a longer half-life than prokaryotic mRNA. Eukaryotic mRNAs are present for many hours, while the standard E.coli bacteria mRNA lasts only 5 seconds.
Pre-mRNAs are initially coated with stabilizing proteins that help protect the pre-mRNA against degradation during mRNA processing in eukaryotes and subsequently exported from the nucleus. The three primary processes of mRNA processing in eukaryotes are the introduction of signalling and stabilizing factors at the 5′ and 3′ ends of the molecule and the elimination of any intervening sequences that do not contain the correct amino acids. In rare instances, the mRNA transcript may be “edited” after transcribed.
mRNA Processing In Eukaryotes
While the pre-mRNA remains being made, the cap 7-methylguanosine is added to the 5′-end of the growing transcript via the 5′-to-5′ phosphate linkage. This moiety helps protect the developing transcript from degrading. In addition, initiation factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes.
5′ cap structure: Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5′ end. The cap protects the 5′ end of the primary RNA transcript from attack by ribonucleases and is recognized by eukaryotic initiation factors involved in assembling the ribosome on the mature mRNA prior to initiating translation. Image Source: https://bio.libretexts.org/
3′ Poly-A Tail
While RNA Polymerase II is still transcribing downstream of the proper end of a gene, the pre-mRNA is cleaved by an endonuclease-containing protein complex between an AAUAAA consensus sequence and a GU-rich sequence. This frees the functional pre-mRNA from the remainder of the transcript, but it remains linked to RNA polymerase.
A protein called poly (A) polymerase (PAP) is part of the same protein complex that cuts the pre-mRNA and then adds a string of around 200 A nucleotides, referred to as “the poly (A) tail that is attached to the 3′-end of the pre-mRNA that has been cleaved. Its poly (A) tail helps protect the mRNA from degrading, assists in the transfer of mature mRNA into the cytoplasm, and plays a role in binding proteins involved in triggering translation.
Poly (A) Polymerase adds a 3′ poly (A) tail to the pre-mRNA: The pre-mRNA is cleaved off the rest of the growing transcript before RNA Polymerase II has stopped transcribing. This cleavage is done by an endonuclease-containing protein complex that binds to an AAUAAA sequence upstream of the cleavage site and to a GU-rich sequence downstream of the cut site. Immediately after the cleavage, Poly (A) Polymerase (PAP), which is also part of the protein complex, catalyzes the addition of up to 200 A nucleotides to the 3′ end of the just-cleaved pre-mRNA.
Eukaryotic genes are composed of protein-coding sequences (ex-on signifies that they’re expressed) and intervening sequences, known as introns (int-ron denotes their intervening function), that could play a role in regulating genes. However, they are taken out of the pre-mRNA processing, and Intron sequences in mRNA cannot encode functional proteins.
Discovery of Introns
Introns were discovered as a shock for researchers in the 1970s who had hoped that pre-mRNAs would define proteins with no further mRNA processing in eukaryotes, just as they seen in prokaryotes. Higher eukaryotes’ genes frequently contain introns or others. These regions might be linked to sequences that regulate the body; the significance of having lots of introns or having very long introns within a gene isn’t clear.
Introns may slow down gene expression as it takes more time to translate pre-mRNAs with many introns. Or, they could be non-functional sequence fragments left due to the fusion of old genes during evolution. This is proven with the evidence that different exons typically encode distinct domains or subunits of proteins. In most cases, intron sequences can be altered without impacting the protein.
Every intron within a pre-mRNA must be eliminated entirely and accurately before the protein synthesis. If the process goes wrong with just one nucleotide, readers of joined exons will shift, and the protein results will be defective. The process of eliminating the introns and reconnecting them is known as splicing. Introns are eliminated and degraded while the pre-mRNA remains inside the nucleus. Splicing is performed using a sequence-specific process, ensuring the removal of introns and exons are joined using the precision of one nucleotide. The splicing process of pre-mRNAs is performed by complexes of proteins and RNA molecules known as spliceosomes.
Pre-mRNA splicing: Pre-mRNA splicing involves the precise removal of introns from the primary RNA transcript. The splicing process is catalysed by large complexes called spliceosomes. Each spliceosome is composed of five subunits called snRNPs. The spliceseome’s actions result in the splicing together of the two exons and the release of the intron in a lariat form. mRNA processing in eukaryotes Source: https://courses.lumenlearning.com
Each spliceosome consists of five snRNPs (for tiny nuclear ribonucleoparticles and is pronounced “snurps”.) Each snRNP is an intricate complex of proteins and an RNA type located within the nucleus, “snRNAs” (small nucleotide RNAs). Spliceosomes detect sequences situated in the 5′ end of the intron since introns are always started with nucleotides GU. They recognize sequences located at the 3′ end of the intron as they end up with nucleotides AG. The spliceosome breaks the sugar phosphate-based backbone of the pre-mRNA at the G, which starts the intron. It then covalently connects the G with an external A nucleotide inside the intron. The spliceosome subsequently connects the 3 ends of the first exon with the 5′-end of the second exon and cleaves the 3′-end of the intron. This results in the splicing together of the two exons and the release of the intron in a lariat form.
Mechanism of pre-mRNA splicing: The snRNPs of the spliceosome were left out of this figure, but it shows the sites within the intron whose interactions are catalysed by the spliceosome. Initially, the conserved G which starts an intron is cleaved from the 3′ end of the exon upstream to it and the G is covalently attached to an internal A within the intron. Then the 3′ end of the just-released exon is joined to the 5′ end of the next exon, cleaving the bond that attaches the 3′ end of the intron to its adjacent exon. This both joins the two exons and removes the intron in lariat form.
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