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RNA polymerase II constructs the primary transcript using a set of four specific ribonucleoside monophosphate residues (adenosine monophosphate (AMP), cytidine monophosphate (CMP), guanosine monophosphate (GMP), and uridine monophosphate (UMP)) that are added continuously to the 3' hydroxyl group on the 3' end of the growing mRNA. [1]
However, DNA and RNA differ in the second major pyrimidine. DNA contains thymine (T) while RNA contains uracil (U). There are some rare cases where thymine does occur in RNA and uracil in DNA. [1] Here are the 4 major ribonucleotides (ribonucleoside 5'-monophosphate) which are the structural units of RNAs.
Initially, the synthesized RNA is a polyphosphate structure. This is why the dephosphorylation is needed, in order to obtain monophosphate by the action of a RNA pyrophosphohydrolase PppH. The transcripts have two parts: the phosphate terminal (P-terminal) and a stem-loop structure as an end.
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 eukaryotes, polyadenylation is part of the process that produces mature mRNA for translation.
RNA polymerase, assisted by one or more general transcription factors, then selects a transcription start site in the transcription bubble, binds to an initiating NTP and an extending NTP (or a short RNA primer and an extending NTP) complementary to the transcription start site sequence, and catalyzes bond formation to yield an initial RNA product.
The process of messenger RNA decapping consists of hydrolysis of the 5' cap structure on the RNA exposing a 5' monophosphate. In eukaryotes, this 5' monophosphate is a substrate for the 5' exonuclease Xrn1 [1] and the mRNA is quickly destroyed. There are many situations which may lead to the removal of the cap, some of which are discussed below.
Transcription of single-stranded RNA from a double-stranded DNA template requires the selection of one strand of the DNA template as the template strand that directly interacts with the nascent RNA due to complementary sequence. The other strand is not copied directly, but necessarily its sequence will be similar to that of the RNA.
RNA integrity can also be analyzed quantitatively comparing the ratio and intensity of 28S RNA to 18S RNA reported in the RNA Integrity Number (RIN) score. [23] Since mRNA is the species of interest and it represents only 3% of its total content, the RNA sample should be treated to remove rRNA and tRNA and tissue-specific RNA transcripts. [23]