Five-prime Cap
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In
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
, the five-prime cap (5′ cap) is a specially altered
nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecul ...
on the
5′ end Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar- ...
of some
primary transcript A primary transcript is the single-stranded ribonucleic acid (RNA) product synthesized by transcription of DNA, and processed to yield various mature RNA products such as mRNAs, tRNAs, and rRNAs. The primary transcripts designated to be mRNAs ...
s such as precursor messenger RNA. This process, known as mRNA capping, is highly regulated and vital in the creation of stable and
mature messenger RNA Mature messenger RNA, often abbreviated as mature mRNA is a eukaryotic RNA transcript that has been spliced and processed and is ready for translation in the course of protein synthesis. Unlike the eukaryotic RNA immediately after transcription ...
able to undergo
translation Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
during
protein synthesis Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside cells, balancing the loss of cellular proteins (via degradation or export) through the production of new proteins. Proteins perform a number of critical ...
.
Mitochondrial A mitochondrion (; ) is an organelle found in the cells of most Eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is us ...
mRNA and
chloroplast A chloroplast () is a type of membrane-bound organelle known as a plastid that conducts photosynthesis mostly in plant and algal cells. The photosynthetic pigment chlorophyll captures the energy from sunlight, converts it, and stores it ...
ic mRNA are not capped.


Structure

In
eukaryotes Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...
, the 5′ cap (cap-0), found on the 5′ end of an mRNA molecule, consists of a
guanine Guanine () (symbol G or Gua) is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine ( uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is c ...
nucleotide connected to mRNA via an unusual 5′ to 5′
triphosphate Polyphosphates are salts or esters of polymeric oxyanions formed from tetrahedral PO4 ( phosphate) structural units linked together by sharing oxygen atoms. Polyphosphates can adopt linear or a cyclic ring structures. In biology, the polyphosph ...
linkage. This guanosine is methylated on the 7 position directly after capping ''in vivo'' by a methyltransferase. It is referred to as a 7-methylguanylate cap, abbreviated m7G. In multicellular eukaryotes and some viruses, further modifications exist, including the methylation of the 2′ hydroxy-groups of the first 2
ribose Ribose is a simple sugar and carbohydrate with molecular formula C5H10O5 and the linear-form composition H−(C=O)−(CHOH)4−H. The naturally-occurring form, , is a component of the ribonucleotides from which RNA is built, and so this compou ...
sugars of the 5′ end of the mRNA. cap-1 has a methylated 2′-hydroxy group on the first ribose sugar, while cap-2 has methylated 2′-hydroxy groups on the first two ribose sugars, shown on the right. The 5′ cap is chemically similar to the
3′ end Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-ri ...
of an RNA molecule (the 5′ carbon of the cap ribose is bonded, and the 3′ unbonded). This provides significant resistance to 5′
exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is t ...
s. Small nuclear RNAs contain unique 5′-caps. Sm-class snRNAs are found with 5′-trimethylguanosine caps, while Lsm-class snRNAs are found with 5′-monomethylphosphate caps. In
bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...
, and potentially also in higher organisms, some RNAs are capped with NAD+, NADH, or 3′-dephospho-coenzyme A. In all organisms, mRNA molecules can be decapped in a process known as messenger RNA decapping.


Capping process

The starting point for capping with 7-methylguanylate is the unaltered 5′ end of an RNA molecule, which terminates at a triphosphate group. This features a final nucleotide followed by three phosphate groups attached to the 5′ carbon. The capping process is initiated before the completion of transcription, as the nascent pre-mRNA is being synthesized. # One of the terminal phosphate groups is removed by RNA triphosphatase, leaving a bisphosphate group (i.e. 5′(ppN) Nsub>n); # GTP is added to the terminal bisphosphate by mRNA guanylyltransferase, losing a
pyrophosphate In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a P–O–P linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate (Na2H2P2O7) and tetrasodium pyrophosphate (Na4P2O7), among o ...
from the GTP substrate in the process. This results in the 5′–5′ triphosphate linkage, producing 5′(Gp)(ppN) Nsub>n; # The 7-nitrogen of guanine is methylated by mRNA (guanine-''N''7-)-methyltransferase, with ''S''-adenosyl-L-methionine being demethylated to produce ''S''-adenosyl-L-homocysteine, resulting in 5′(m7Gp)(ppN) Nsub>n (cap-0); # Cap-adjacent modifications can occur, normally to the first and second nucleotides, producing up to 5′(m7Gp)(ppN*)(pN*) Nsub>n (cap-1 and cap-2); # If the nearest cap-adjacent nucleotide is 2′-''O''-ribose methyl-adenosine (i.e. 5′(m7Gp)(ppAm) Nsub>n), it can be further methylated at the N6 methyl position to form ''N''6-methyladenosine, resulting in 5′(m7Gp)(ppm6Am) Nsub>n. The mechanism of capping with NAD+, NADH, or 3′-dephospho-coenzyme A is different. Capping with NAD+, NADH, or 3′-dephospho-coenzyme A is accomplished through an "ab initio capping mechanism," in which NAD+, NADH, or 3′-desphospho-coenzyme A serves as a "non-canonical initiating nucleotide" (NCIN) for transcription initiation by
RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...
and thereby directly is incorporated into the RNA product. Both bacterial RNA polymerase and eukaryotic
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNAP enzymes found in the nucleus of eukary ...
are able to carry out this "ab initio capping mechanism".


Targeting

For capping with 7-methylguanylate, the capping enzyme complex (CEC) binds to
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNAP enzymes found in the nucleus of eukary ...
before transcription starts. As soon as the 5′ end of the new transcript emerges from RNA polymerase II, the CEC carries out the capping process (this kind of mechanism ensures capping, as with
polyadenylation Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA (mRNA). The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eu ...
). The enzymes for capping can only bind to
RNA polymerase II RNA polymerase II (RNAP II and Pol II) is a multiprotein complex that transcribes DNA into precursors of messenger RNA (mRNA) and most small nuclear RNA (snRNA) and microRNA. It is one of the three RNAP enzymes found in the nucleus of eukary ...
, ensuring specificity to only these transcripts, which are almost entirely mRNA. Capping with NAD+, NADH, or 3′-dephospho-coenzyme A is targeted by promoter sequence. Capping with NAD+, NADH, or 3′-dephospho-coenzyme A occurs only at promoters that have certain sequences at and immediately upstream of the transcription start site and therefore occurs only for RNAs synthesized from certain promoters.


Function

The 5′ cap has four main functions: # Regulation of nuclear export; # Prevention of degradation by
exonuclease Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is t ...
s; # Promotion of translation (see ribosome and
translation Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
); # Promotion of 5′ proximal intron excision. Nuclear export of RNA is regulated by the
cap binding complex The 5' cap of eukaryotic messenger RNA is bound at all times by various cap-binding complexes (CBCs). Nuclear cap-binding complex In the nucleus, freshly transcribed mRNA molecules are bound on the 5' cap by the nuclear cap-binding complex of C ...
(CBC), which binds exclusively to 7-methylguanylate-capped RNA. The CBC is then recognized by the nuclear pore complex and exported. Once in the cytoplasm after the pioneer round of translation, the CBC is replaced by the translation factors
eIF4E Eukaryotic translation initiation factor 4E, also known as eIF4E, is a protein that in humans is encoded by the ''EIF4E'' gene. Structure and function Most eukaryotic cellular mRNAs are blocked at their 5'-ends with the 7-methyl- guanosine ...
and
eIF4G Eukaryotic translation initiation factor 4 G (eIF4G) is a protein involved in eukaryotic translation initiation and is a component of the eIF4F cap-binding complex. Orthologs of eIF4G have been studied in multiple species, including humans, yeast ...
of the eIF4F complex. This complex is then recognized by other translation initiation machinery including the ribosome. Capping with 7-methylguanylate prevents 5′ degradation in two ways. First, degradation of the mRNA by 5′ exonucleases is prevented (as mentioned above) by functionally looking like a 3′ end. Second, the CBC and eIF4E/eIF4G block the access of decapping enzymes to the cap. This increases the
half-life Half-life (symbol ) is the time required for a quantity (of substance) to reduce to half of its initial value. The term is commonly used in nuclear physics to describe how quickly unstable atoms undergo radioactive decay or how long stable at ...
of the mRNA, essential in eukaryotes as the export and translation processes take significant time. Decapping of a 7-methylguanylate-capped mRNA is catalyzed by the decapping complex made up of at least Dcp1 and Dcp2, which must compete with eIF4E to bind the cap. Thus the 7-methylguanylate cap is a marker of an actively translating mRNA and is used by cells to regulate mRNA half-lives in response to new stimuli. Undesirable mRNAs are sent to P-bodies for temporary storage or decapping, the details of which are still being resolved. The mechanism of 5′ proximal intron excision promotion is not well understood, but the 7-methylguanylate cap appears to loop around and interact with the
spliceosome A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs ( snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to sp ...
in the splicing process, promoting intron excision.


See also

* Messenger RNA decapping * Eukaryotic initiation factor 4F (eIF4F) * Cap analysis gene expression


References


External links

* {{Post transcriptional modification RNA