In this article we will discuss about the processing of mRNA in prokaryotes and eukaryotes.
In prokaryotes, there is a little or no processing of mRNA transcripts. Prokaryotic mRNA is degraded very rapidly from 5′ end. Therefore, to rescue from degradation it is translated before being finally transcribed. Ribosomes assemble on unfinished mRNA and first cistron (protein coding region) can be translated very soon. The internal cistrons are partially protected by stem-loop structure formed at 5′- and 3′- ends.
The eukaryotic RNA Pol II transcribes different genes from snRNA genes and forms a collection of products which is known as heterogenous nuclear RNA (hnRNA). The pre-mRNA transcripts undergo processing to form mature mRNAs.
Processing events are briefly described herewith:
i. The hnRNP:
RNA Pol II synthesizes the hnRNA, which is mainly pre-mRNA. Soon after synthesis hnRNA is covered by three important hnRNP proteins (e.g. A, B and C proteins) to form heterogeneous nuclear ribonucleoprotein (hnRNP) particles. They contain three copies of three tetramers and about 600-700 nucleotides of hnRNA. Possibly the hnRNP proteins keep the hnRNA in a single stranded form and help in various events of RNA processing.
ii. The snRNP Particles:
There are many uracil-rich snRNA molecules denoted by U1, U2, etc. Most of them are transcribed by RNA Pol II which complex with specific proteins and form snRNPs. The most abundant are involved in pre-mRNA splicing- U1, U2, U4, U5 and U6.
A majority of them is involved in determining the methylation sites of pre-rRNA and located in nucleolus. The major snRNAs containing the sequence 5′-RA(U)n GR-3′ bind with eight common proteins in the cytoplasm and become hyper-methylated. Thereafter, these are transported back into the nucleus.
Soon after synthesis of about 25 nucleotide-long mRNA chain by RNA pol II, the 5′-end is chemically modified by the addition of a 7-methylguanosine (m7G) residue. This 5′ modification is called a CAP which is done by addition of a GMP nucleotide to the new transcript.
It is added (by an enzyme mRNA guanyltransferase) in a reverse polarity (5′ to 5′ triphosphate bridge) as compared with the normal linkage. This cap acts as a barrier to 5′-exonuclease attack, but it promotes splicing transport and translation processes.
iv. 3′ Cleavage and Polyadenylation:
The mature 3′-end in most pre-mRNA molecules is generated by cleavage at polyadenylation. For cleavage and polyadenylation reaction specific sequences are present in DNA and its pre-mRNA transcript which consists of 5′-AAUAAA-3′ that provides polyadenylation signal.
It is followed by a 5′-YA-3′ and GU-rich sequence (Fig. 10.12). Collectively these sequences are called polyadenylation site. Then about 250 nucleotides long poly (A) tail is added by poly (A) polymerase (PAP) and mature mRNA is generated.
The eukaryotic pre-mRNA possesses introns (intervening sequencing) that interrupt the exons (the coding regions). The pre-mRNA is cut, introns are spliced out (removed) and two flanking exons are joined together. This process is called splicing. This event takes place in nucleus before transport of mature mRNA in cytoplasm. For splicing, introns require to have a 5′-GU, an AG-3′ and a branch-point sequence.
The introns are removed in a two-step reaction as a tailed circular molecule called lariat which is degraded later on (Fig. 10.13). The splicing process is catalysed by the snRNPs (U1, U2, U4, U5 and U6) and the other splicing factors.
The complex of snRNPs and pre-mRNA holds the upstream and downstream exons close together and the looping out introns is called splisosome. Reaction for cleavage of introns and ligation of exons take place inside the splisosome releasing the introns as a lariat.
vi. Pre-mRNA Methylation:
In pre-mRNA (containing the sequence 5′-RRACX-3′ where R= purine) a small percentage of A residues becomes methylated at the N6 position.