Messenger RNA (mRNA) carries genetic instructions from the DNA in the nucleus to the protein-synthesizing machinery in the cytoplasm. In eukaryotic cells, the initial RNA transcript (pre-mRNA) must undergo modifications to become a functional, mature mRNA molecule. These modifications protect the transcript and prepare it for translation, where the genetic code is read to build a protein. The first and most significant alteration is the addition of a specialized structure at the 5′ end of the newly synthesized strand, known as the 5′ cap.
Defining the 5′ Cap: Structure and Chemistry
The 5′ cap is a chemically distinct modification formed by a single methylated guanine nucleotide, called 7-methylguanosine (m7G). This m7G molecule attaches to the first transcribed nucleotide of the mRNA via an unusual chemical bond.
Standard RNA nucleotides are connected by 3′-5′ phosphodiester bonds. In contrast, the 5′ cap is joined to the mRNA via a 5′-5′ triphosphate linkage, connecting two 5′ carbons with three phosphate groups. This “reverse linkage” is a defining characteristic of the cap and is responsible for many of its functions.
The modification uses Guanosine Triphosphate (GTP), which provides the guanosine base and the triphosphate bridge. The simplest form, Cap 0, features only the m7G linked to the first nucleotide. In higher organisms, the ribose sugars of the first or second nucleotides may also be methylated at the 2′-O position, creating Cap 1 and Cap 2 structures. These additional methylations help the cell distinguish its own transcripts from foreign RNA.
The Co-Transcriptional Capping Mechanism
The 5′ cap attachment is tightly coordinated with mRNA synthesis, a process called co-transcriptional capping. This modification begins while the RNA is still being produced by RNA Polymerase II (Pol II), typically when the nascent transcript is only 20 to 30 nucleotides long.
The capping process involves a sequential three-step enzymatic reaction. First, an RNA triphosphatase removes one phosphate group from the 5′ end, leaving a diphosphate. Next, a guanylyltransferase adds a guanosine monophosphate (GMP) from a GTP molecule, forming the 5′-5′ triphosphate bridge. Finally, a methyltransferase adds a methyl group to the guanine base at the N7 position, completing the m7G cap.
The capping enzymes are recruited and positioned near the transcription site by the C-Terminal Domain (CTD) of RNA Pol II. The CTD’s phosphorylation status acts as a signal, ensuring the enzymes are recruited precisely when the transcript begins to emerge.
Ensuring mRNA Longevity and Stability
A primary function of the 5′ cap is protecting the mRNA against molecular degradation. RNA transcripts are constantly under threat from nucleases, enzymes that break down nucleic acids, particularly 5′ exonucleases that attack the exposed 5′ end.
The m7G cap acts as a shield due to its unique chemical structure. The 5′-5′ reverse linkage blocks the recognition site for 5′ exonucleases, preventing premature breakdown and increasing the molecule’s half-life within the cell.
Immediately after formation, the cap associates with the Cap-Binding Complex (CBC) in the nucleus. This complex, composed of proteins CBP20 and CBP80, prevents the cap from being removed by decapping enzymes, which would initiate rapid degradation.
Facilitating Protein Production and Nuclear Export
The 5′ cap also serves as a crucial molecular flag directing the mRNA through the final stages of gene expression. The nuclear Cap-Binding Complex (CBC) binds the cap immediately upon formation. The CBC facilitates the transport of the mature mRNA through nuclear pores and into the cytoplasm, where protein synthesis occurs.
Once in the cytoplasm, the nuclear CBC is replaced by proteins involved in translation initiation. The cap is the primary recognition site for eukaryotic initiation factor 4E (eIF4E), a component of the larger eIF4F complex. Binding of eIF4E to the cap is one of the first and most regulated steps in protein synthesis.
The eIF4F complex recruits the small 40S ribosomal subunit to the mRNA, acting as a landing pad. This cap-dependent recruitment is a rate-limiting step for translation in eukaryotic cells. By serving as this binding site, the m7G cap ensures the protein-building machinery correctly identifies the genetic instructions and begins translation efficiently.