In eukaryotes, post-transcriptional modifications are essential steps in maturing the newly synthesized RNA, making it ready for translation. Two key modifications—the 5' cap and the 3' poly(A) tail—serve protective and regulatory roles in mRNA stability and function. Together, the 5' cap and the poly(A) tail stabilize and prepare the mRNA for translation, ensuring it is protected and efficiently recognized by the cell's protein-making machinery.

Process of 5' Capping in Eukaryotic mRNA

The 5' capping process is essential in preparing mRNA for translation and protecting it from degradation. This stepwise breakdown will make it easier to understand each part of the capping process.

Step 1: RNA Polymerase Begins Transcription

• As transcription begins, RNA polymerase II synthesizes a new RNA molecule by reading the DNA template. After about 20-30 nucleotides have been added, the capping process is triggered.

Step 2: Removal of the 5' Phosphate Group

• The first modification involves the removal of one phosphate group from the 5' end of the RNA. This is done by an enzyme called RNA triphosphatase, which converts the 5' triphosphate end of the RNA into a 5' diphosphate end.

Step 3: Addition of a Guanine Base

• An enzyme called guanylyltransferase then attaches a molecule of guanosine monophosphate (GMP) to the 5' diphosphate end through an unusual 5' to 5' triphosphate linkage. This added guanine creates what’s called a "cap" on the RNA’s 5' end.

Step 4: Methylation of Guanine (Cap 0 Structure)

• Next, a methyl group (CH₃) is added to the guanine base by the enzyme guanine-7-methyltransferase. This modification creates a "Cap 0" structure, where the 5' cap now has a methylated guanine base.

Step 5: Further Methylation (Cap 1 and Cap 2 Structures)

• In some eukaryotic RNAs, additional methyl groups are added to the ribose sugars of the first and second nucleotides adjacent to the cap. This results in "Cap 1" (one additional methylation) or "Cap 2" (two additional methylations) structures.

  • Cap 1: Methyl group is added to the 2' hydroxyl group of the ribose sugar on the first nucleotide after the cap.

  • Cap 2: Another methyl group is added to the ribose sugar of the second nucleotide.

Step 6: Cap-Binding Complex (CBC) Attachment

• Finally, a Cap-Binding Complex (CBC) attaches to the 5' cap structure. This protein complex helps transport the mRNA from the nucleus to the cytoplasm and also facilitates recognition by the ribosome for translation.

Summary of Functions of the 5' Cap

• Protection: The cap protects mRNA from degradation by exonucleases (enzymes that degrade RNA).

• Transport: The cap-binding complex aids in exporting the mRNA from the nucleus to the cytoplasm.

• Translation Initiation: The 5' cap structure is recognized by the ribosome, which is crucial for initiating translation.

This capping process is a critical step in mRNA processing, ensuring that mRNA is stable and properly recognized for protein synthesis.

Process of Poly(A) Tail Formation in Eukaryotic mRNA

The addition of a poly(A) tail to the 3' end of an mRNA molecule is essential for mRNA stability, export, and translation efficiency. Here’s how the process unfolds:

Step 1: Transcription Reaches the Polyadenylation Signal

• As RNA polymerase II transcribes the DNA, it eventually reaches a specific sequence on the mRNA known as the polyadenylation signal sequence (usually "AAUAAA" or a similar sequence).

• This sequence signals that the end of the mRNA transcript is near and initiates the polyadenylation process.

Step 2: Cleavage of the mRNA Transcript

• A complex of proteins, including CPSF (Cleavage and Polyadenylation Specificity Factor) and CstF (Cleavage Stimulatory Factor), recognizes and binds to the polyadenylation signal sequence.

• CPSF and CstF recruit additional proteins to form the cleavage and polyadenylation complex.

• This complex then cleaves the mRNA a few nucleotides downstream of the polyadenylation signal, creating a new 3' end on the mRNA.

Step 3: Addition of Adenine Nucleotides (Polyadenylation)

• After cleavage, an enzyme called poly(A) polymerase (PAP) begins to add a chain of adenine nucleotides (A's) to the 3' end of the mRNA, forming the poly(A) tail.

• Initially, around 10-20 adenines are added slowly.

Step 4: Rapid Extension of the Poly(A) Tail

• Once the initial adenines are in place, PABP (Poly(A)-Binding Protein) binds to the new poly(A) tail, protecting it and stabilizing it.

• With PABP in place, poly(A) polymerase can rapidly add additional adenines, extending the poly(A) tail to about 100-250 nucleotides in length.

Step 5: Completion and Stabilization of the Poly(A) Tail

• The fully-formed poly(A) tail, bound by multiple PABPs, protects the mRNA from degradation by enzymes, ensuring its stability.

• PABP also plays a role in transporting the mRNA from the nucleus to the cytoplasm and in initiating translation.

Summary of Functions of the Poly(A) Tail

• Protection: The poly(A) tail protects the mRNA from exonucleases, which could degrade the RNA from the 3' end.

• Transport: The tail, with the help of PABPs, facilitates the export of mRNA from the nucleus to the cytoplasm.

• Translation Efficiency: The poly(A) tail, along with PABP, interacts with translation initiation factors, enhancing the mRNA’s efficiency in protein synthesis.

The poly(A) tail is thus a vital modification, supporting the mRNA throughout its journey from transcription to translation.