Messenger RNAs (mRNAs):
Among the different species of RNAs, it is only mRNAs that are linearly related to DNA and to polypeptide chain as an intermediary. It is this species of RNA that carries encoded message from the master molecule in the form of codons. It is these molecules that translate by decoding the information into polypeptide chains. It is ultimately the protein, having a unique structural and functional properties determine the structure and function of the cell that is why proteins are deemed to be molecular demy-gods. Proteins are the quintessence of the gene function at molecular level. Here the triple or tripartite relationship, between linear DNA, linear mRNA and linear polypeptide chain (in the form of nucleotides sequence and amino acid sequence respectively), is referred to as co-linearity. Two important functions that occur between the gene and the polypeptide chain, they are transcription and translation, which are separated in space and time.
General Features of Prokaryotic mRNAs:
· Prokaryotic mRNAs are made up of a polynucleotide chains of a specific sequence endowed with 5’ to 3’ polarity.
· Though it is linear, it can exhibit several but short, secondary structures.
· Size of the mRNAs is more or less proportional to the gene from which it generates.
· The polypeptide chain it produces is directly proportional to the coding size of the mRNA. Longer the protein longer is the mRNA and vice versa.
· Average size of mRNA in bacterial systems is 1000 ntds to 1500.
· All mRNAs consists of a 5’ non-coding, called 5’UTR, called leader sequence, whose length and sequence varies from one species of mRNA to the other.
Polycistronic mRNA with spacer between cistrons
· The leader sequence in every mRNA contains an all-important structured sequence called Shine-Delgarno sequence, seven to nine nucleotides upstream of the start codon AUG.
· All mRNAs also contain a non-coding 3’ terminal sequence, whose structure; size and function vary among the members of each species of mRNAs.
· Every functional mRNA consists of coding region, which lies between 5’ leader segment and 3’ terminal non-coding sequence.
· The coding region starts from the initiator codon AUG (mostly) or GUG (rarely) and ends in a terminator codon like UAG, UGA or UAA.
· The region between initiator codon and terminator codon is often called open reading frame (ORF). Within ORF there is no scope for any codon to be non-coding, but there can be missense codon. If the reading frame changes by one or more nucleotides by insertion or deletion the overall meaning of the message changes.
· The ORF refers to the region between initiator codon and terminator codon, but the initiator codon should be in proper sequence context for the initiating translation.
· In the 5’ region there may be several initiator codons (i.e. AUGs, very rarely GUGs), but only one of the AUGs will be in right context, in the sense the sequences around the Initiator codon is important for correct and proper initiation of translation. If it initiates at wrong AUG translation will be terminated for it certainly encounters a terminator codon.
· In the case of prokaryotes, the coding region is split consisting of cistrons with initiating and terminator sequences. In between two such coding regions one finds a short spacer region, hence the name polycistronic.
· This spacer region is used for the translation termination of the cistron and again initiates at the next cistron, the size of the inter-cistronic space varies, but mostly very short.
In prokaryotes transcription and translation are coupled.
· Half-life of mRNAs is very short ranging between 2-5 minutes.
· All mRNAs are single stranded polynucleotide chains with 5ŕ3’ polarity and mostly monocistronic.
· Average size of eukaryotic mRNAs is 1500 to 2000 nucleotides. Smaller mRNAs are found in mammalian systems such as
mRNAs for polypeptide hormones like Oxytocin, Glucagons and few such proteins. The longest mRNAs found are of proteins such as Dystrophin, Apolipoprotein, Fibronectin and Titin and few others.
· The open reading frame (ORF) starts with an initiator codon and ends in a terminator codon.
· Upstream of the initiator codon pre-mRNAs contain non translatable sequences called leader or UTR. Similarly at the 3’ end after TER codons, most of the mRNAs contain another 3’UTR trailer sequence, which vary in length and sequence.
· Secondary structure in leader and 3’ terminal regions play an important role in the regulation of mRNA stability or and its ability and efficiency to translate. The said 3’ sequences are used for making the mRNA inactive to be stored for a long time. Unfertilized eggs, developing oocytes and plant seeds do store mRNA as informosomes.
· The initiator codon should be in proper context for proper initiation. In eukaryotes, ACC AUG G or A / G CC AUG G sequence is the most important sequence context, without which translation initiation fails, even if initiates, it will be terminated shortly for it encounters a terminator codon. It is this sequence that acts at the time translation initiation is considered as the start of ORF .
· The said sequence is called Kozak sequence.
· In addition to initiator sequences, positioning the Kozak sequence in right context, the succeeding sequences have to be in the order three and should end in a terminator codon, it is only then such sequence is called open reading frame. Other wise, if one finds one or more initiator codons in 5’ terminal region, one cannot consider it as start of the reading frame. Any changes in this module make the mRNA useless.
· At 5’ end the mRNA is added with 7’ methyl Guanine covalently to the first nucleotide, invariably to ‘A’ by an unusual 5’ P to 5’P linkage (7’CH3 G5’-P-P-P5’A---), called ‘cap-O’. Subsequent addition of more methyl groups to 7’A or to 2’ OH groups of ribose sugars in succeeding nucleotides make them to be called, cap-1, cap-2 etc.
· Only eukaryotic mRNAs contain cap structures and few non coding RNAs such as Sn RNAs, sno RNAs and few viral RNA genomes have such structures.
· Caps increase the efficiency of translational initiation of mRNA.
· Cells do contain specific CAP binding proteins.
· Only capped mRNAs are transported out of nuclei.
· At the 3’ end, mRNAs are added with a 200-to 250-nucleotide long poly (A) tail. However EK mitochondrial mRNAs ex. From yeast and Neurospora, the poly (A) tail is just 50 ntds long.
· A poly-A signal with AAAUAA and G/U rich sequence is used for cleaving the extended 3’ end of mRNA at 28-30 ntds from the signal, then poly-As are added to the cut end.
· Synthesis and translation are separated in space and time. It takes nearly 30 to 45 minutes for the functional mRNAs to appear in cytoplasm after its synthesis.
· All mRNAs are synthesized as long precursor RNAs called heterogeneous nuclear RNAs (hnRNAs), which are also called pre-mRNAs. Because of the heterogeneity in size and characters of the mRNAs produced, they are also called hn RNAs.
· The hn-RNAs, in most of the cases, longer than their counter part cytoplasmic functional mRNAs.
· They (hnRNAs) contain several segments of non-coding sequences, called Introns or intervening or interspersed sequences among coding regions called Exons.
· The number of introns and the size of each introns and the position of introns vary from one species of mRNA to the other.
· Introns are removed and specific Exons are joined in a sequence by a process called splicing.
· Only spliced mRNAs are transported out of the nucleus.
· The same mRNA produced in different tissues can be spliced in different ways, by what is called alternate splicing to produce different polypeptides in stage specific or tissue specific manner, depending on the needs.
· Some mRNAs are edited in tissue specific manner by specific enzymes or by Editosomes.
· Many systems perform trans-splicing resulting in all mRNAs having the same leader sequence. Here leader sequences and mRNA or pre-mRNAs are synthesized separately, and then they are processed to produce transpliced mRNAs.
· Majority of the mRNAs exhibit rapid turnover, yet their half-life is more than 10 to 20 minutes; some have long half-lives up to 120 days.
· Some mRNAs associated with a variety of mRNPs remain untranslated for quite a period of time, such mRNAs are called informosomes. They are activated differentially, when required, with specific signals.
· In eukaryotes (Eks)) certain species of mRNAs are positioned in specific locations inside the cell, ex. Segmentation mRNAs in developing Drosophila larvae, mRNAs for egg polarity fixation, and Actins in developing Myeloblast cells.
· Such localization is made possible by their poly (A) tails where such mRNAs are actually held by cross-linking to Actin filament network. Microtrabacular network of Actin like filaments of small sizes may have a role?
· Once processed mRNAs are coated with specific mRNPs and transported out of the nucleus.
· For transportation, mRNAs must have a cap structure, with out which they cannot be transported out of the nucleus. In addition specific proteins are required for transportation; otherwise they remain within the nucleus.
At 5’UTR region it contains stem loop structures that have regulatory functions. Some of the mRNA contain internal ribose entru elements called IREs.
The 3’UTR contain many sequence elements such as Antisense binding sequences, regulatory stem loops, ARE sequences, mRNA localization sequences and cytoplasmic poly adenylation elements.
Most of the hn RNPs are heterogeneous in character and molecular weight. Based on their mobility in the gel and affinity to poly (A), poly (U), poly(C), poly (G) or poly (T) nucleotide chains, a large number of proteins, have been isolated and designated as hn RNP-A to hn-RNP-U and so on. Smallest of the proteins is A1, A2 and the highest Mol.wt proteins are U1, U2 proteins. The molecular mass of them ranges from 34 KD to 120kds. Most of the proteins have RNA binding motifs and also protein-protein binding motifs.
A list of proteins hnRNPs (not all are included):
A1A2, B1B2, C1C2, D1D2, E (4), F (2), G, H (2-3), I (2), J, K (3), L (2), M (4), N, P (2), Q (2), R (2), S2, T1, U1 and U2. Mol.wt of A to C range about 30-40kd, D to G 45-48kd, J, H, I 55-58kd, L, M, Q, P, N =68KD, T is95kd, R is 75kd, K is 66kd and U is about 116kd.
· Almost all hnRNPs are derived from normal and alternate splicing. Among them, there are core proteins organized into 40s complexes, invariably they are made up of A1, A2, B1, B2, C1, C2 and the rest of them are grouped as D1 to U1 etc; the latter are called accessory proteins.
· The proteins have RNA binding motifs called RNP motifs, made up of beta strands. Some of the sequences found in proteins and used for RNA binding are RGG box, KH motifs, and SR motifs. Their versatile nature makes them ideal candidates for them to bind to hnRNA or pre-RNAs and prevents the RNAs from the formation of unwanted sec.structures.
· In addition protein-RNA binding and protein-protein interactions bring pre-RNA into focus for modifications.
· Several of hnRNP proteins that bind to pre mRNA and also to processed mRNA have been identified and isolated. Core protein complexes of 200 A^o size uniformly cover the pre-mRNA.
· As soon as an hn RNA is produced or being produces, it is covered with a variety of proteins, among them are SnRNA and snRNP complexes, for they are involved in splicing.
· Processed mRNAs are also bound by a variety of proteins called mRNPs, and some of them may be involved in transport of mRNAs out of Nucleus. Inexplicably, some proteins move out of nucleus in association with mRNAs, and they are displaced with cytosolic mRNPs; and displaced nuclear mRNPs return to nucleus, ex. Poly (A) binding protein PAB-II. Similarly a viral protein REV facilitates HIV RNA to move out of the nucleus; once mRNAs are transported out the nucleus the Rev Protein returns to the nucleus.
· In developing Oocyte of Xenopus, a large number of mRNAs produced are processed and transported into cytoplasm where they remain untranslated and stored in the egg. Such mRNAs covered with a variety of mRNPs, such threads are called informosomes. The kinds of proteins bind to mRNA are species specific, some are cell specific and the rest of them are general.
· Some of the mRNAs stored as informosomes have shortened poly-(A) tail (20As). Fertilization provides signals, which make several of the mRNAs, specifically get activated. At this point of time hitherto inactive mRNAs are added with poly- (A) and such mRNAs are translated.
· Even dry seeds, with 6-8% of water, having inactive embryos, on imbibing water to full capacity (80-90%), several of the stored informosomal mRNAs become active and translate efficiently. This increase in translation is not accompanied with
new synthesis of mRNAs.
Even tissues, which are kept in dark and etiolated, when, exposed to light or hormones or both, mRNAs are activated and they are translated. Such mRNAs contain CPE sequences bound by CPEB which in turn bound by Maskins, thus the mRNAs are rendered inactive.