Messenger RNAs (mRNAs):
Jacques Monod and François Jacob, were the first to suggest mRNA acts between the Gene and the polypeptide. Then Sidney Brenner and Mathew Meselson discovered mRNAs (1961). 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 the information by decoding it 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 work-horses. Proteins are the quintessence of the gene function at molecular level. DNA is considered as the master molecule, but without protein it has no function and without DNA protein has no existence.
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 a gene and the polypeptide chain are transcription and translation, which are separated in space and time.
Translation is the process cells use to synthesize polypeptides using the codon sequence in the mRNA as a template. mRNA; In the bacterial cell, several related polypeptide sequences are carried on the same mRNA template. Each sequence in the mRNA strand has its own ribosome binding site, start and stop codons at the end. Since transcription takes place in the cytoplasm, translation can begin even before transcription is complete. A cistron starts with AUG and ends in UAA. http://www.quora.com/;http://academic.pgcc.edu/;http://csls-text.c.u-tokyo.ac.jp/
Eukaryote mRNA; this kind of mRNA one finds rarely; most of the time the coding region is split into Introns and Exons; http://lh5.ggpht.com/
Each of the cistrons has its own SD sequence and Ter sequence at the end of the cistron; Z cistron starts with AUG and ends in UAA; intercistronic spacer-AAAUCAA- The Y cistron starts with AUG and ends in UAA-intercistronic Z & Y spacer-UAACGGAGUGAUC- and A cistron starts with UUG ends in UAA. http://www.quora.com/; ;http://lh5.ggpht.com/-ijk9yxnlLuU
General Features of PK mRNAs:
Polycistronic mRNA with spacer between cistrons: The mRNA transcript is of Lac Z operon; between Z cistron and Y cistron there is spacer consisting S/D sequence. Each of the cistrons end in a terminator codon and the next cistron has initiator codon AUG or GUG. At the end of the cistron the ribosomes dissociates and reassociate at the next cistron using S/D sequence and starts translation of the next cistron.mol-biol4masters-copy from web
AUG , as translation ends the ribosomes next initiates the next cistron
In prokaryotes transcription and translation events are coupled. Half-life of mRNAs is very short ranging between 2-5 minutes. Most of prokaryotic mRNAs don’t have poly-A tail, even if they have the tail will be 15-20ntds long. This poly-A adenylation induces degradation of mRNAs, in fact it is added during degradation.
Only some mRNA from Yersinia contain bicistronic and monocistronic mRNAs for Major Cold Shock protein (MCSP) induced by cold shock. Bacterial and yeast mRNA lack introns.
In plastids (chloroplasts), primary transcripts undergo a complex series of mRNA maturation steps. These include processing of the 5′ and 3′ ends (RNA trimming), intron splicing, RNA editing, and cleavage of polycistronic precursor transcripts into monocistronic or oligo cistronic mRNAs (RNA cutting). On the other hand, chloroplast mRNA generated, most of them are polycistronic, but for translation they have to be cut to generate monocistronic mRNAs,( FiZhou, Daniel Karcher, Ralph Bock;2007). They have retained their ancestral characters. Plastids’ 5′ and 3′ end processing is catalyzed by nucleus-encoded prokaryotic-type ribonucleases. Whereas 5′ end maturation is catalyzed primarily by endoribonucleases, 3′ end formation is mediated by the concerted action of endoribonucleases and 3′→5′ exoribonucleases. Stem–loop-type RNA secondary structures within the 5′ and 3′ untranslated regions (UTRs) of plastid messenger RNAs provide important recognition elements for RNA processing enzymes, and, in addition, can serve as protective elements preventing rapid RNA degradation (Barkan and Goldschmidt-Clermont, 2000; Mayfield et al., 1995; Monde et al., 2000; Stern and Gruissem, 1987). One of the exceptions is the psbE operon, which comprises four small genes for polypeptides of photosystem II (psbE, psbF, psbL and psbJ;
(A) Transcription initiation of a gene cluster occurs from multiple promoters (bent arrow) upstream of open reading frames (ORFs) or within ORFs. Together with inefficient transcription termination, this setup generates numerous precursor transcripts that can include complete or incomplete ORFs. Introns and RNA stem–loop structures are depicted as light black rectangles and hairpins, respectively. (B) Precursor transcripts are processed by a combination of exo- and endo-ribonucleases. The precursor transcripts also can be polyadenylated by the addition of a Poly(A)-tail at the 3′-end of the transcripts. The sequence-specific RNA-binding proteins define functional RNAs followed by ribonuclease digestion. Introns and incomplete ORFs without sequence-specific RNA-binding proteins protection were digested by exo- or endo-ribonucleases. (C) RNA processing produces a pool of functional RNAs. http://www.nature.com/
Mitochondrial mRNAs are polycistronic, one H strand transcribes and the other is L strand transcribes. These long transcripts are cleaved to liberate tRNAs found in between and monocistronic mRNAs are released, sometimes the terminator codon of one cistron overlaps with the initiator codon of the next cistron. The released monocistronic mRNA are immediately polyadenylated up to ~50 (A) s.
Circularization provides easy way for ribosomes as they finish, they start another round of translation- this is a time programmed and efficient process. Some viral mRNA and some viral RNA genomes also show circularization.
5’- 7’CH3 Gmp (Cap)-http://www.studyblue.com/
topsy.fr/hashtag.php;mol-biol4 masters (copy)
(35%), in humans which increase the expression.
Kozak sequence is a consensus sequence (A/G)CCATGG) found adjoining the initiator codon AUG in eukaryotic mRNA, Harhay GP, et al ;http://openi.nlm.nih.gov/
Consensus RBS Sequences. The +1 A is the first base of the AUG initiator codon (shaded) responsible for binding of fMet-tRNAfMet. The underline indicates the ribosomal binding site sequence, which is required for efficient translation; http://themedicalbiochemistrypage.org/
Histone mRNAs lack Poly-A tails, but contain stem loop structure at 3’ end. Some nc RNA like Lnc and Xist RNA too contain poly-A tail. Some histone mRNAs synthesized in non-cell cycle dependent stages contain poly-A tails. But yeast histone mRNA are poly-Adenylated?
Only capped mRNAs with poly-A tail and spliced ones are transported out of nuclei. But histone mRNA does not contain Poly-A tail, but the length varies. Its 3’ end is also processed to make it functional.
· Synthesis and translation are separated in “time and space”. 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 hnRNA.
· 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.
· More than ~1000 intron less mRNAs are found in human genome transcripts, ex. Histone mRNAs, IFN alpha and IFN-beta mRNAs and many more lack any introns.
· 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 alternative splicing to produce different polypeptides in stage specific or tissue specific or cell type manner, depending on the needs.
· Alternative polyadenylation-In some species a single polycistronic Pre mRNAs contains polyadenylation sites at different positions, thus it produces different mRNAs but its 5’ region remains the same.
· 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 Trans-spliced 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 or stored mRNAs. They are activated differentially, when required, with specific signals.
· mRNP code is not unique to eukaryotic mRNAs, it is also prevalent in bacterial mRNAs.
Different proteins assemble on a given mRNA to form a ribonucleoprotein complex (mRNP), the composition of which changes dynamically, depending on the cellular context. The combinatorial control of associated regulatory, scaffolding and accessory proteins ultimately determines fate of the mRNA ("mRNP code"); http://surrey.ac.uk/ ,k.a.gerber
· In eukaryotes (Eks) certain species of mRNAs are positioned in specific locations inside the cell, ex. positioning of mRNAs in developing segments of Drosophila larvae, mRNAs for egg polarity fixation, and Actins in developing Myeloblast cells. Ex. Oskar and Nano (Posterior), Bicoid (anterior); Staufen a dsRNA binding protein is involved in immobilization of Bicoid RNA at anterior end, Gurken (anterior-dorsal) mRNAs.
· Such localization is made possible by ‘zip code’ or ‘RNA transport signal sequences (21ntds)’ to which specific proteins bind and then they are transported on microtubule tracts using Kinesin or Dyneins motor proteins. In some their poly-(A) tails are actually held by cross-linking to Actin filament network. Microtrabacular network of Actin like filaments of small sizes may have a role? Actin mediated motor proteins are Myosins.
· Some mRNAs contain quadruplex RNA structure in 5’UTR.
· Some eukaryotic mRNAs are also known to contain several types of repeat in the untranslated regions, including short interspersed elements (SINEs) such as Alu elements, long interspersed elements (LINEs), Minisatellite and microsatellites. In human mRNAs, repeats elements are found in about 12% of 5' UTRs and 36% of 3' UTRs. A lower repeat abundance is observed in other taxa, including other mammals.
· Processed mRNAs are bound by 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.
mRNA processing, 5’ capping; https://www.boundless.com
At 5’UTR region some mRNAs contains stem loop structures that have regulatory functions. Some of the mRNAs contain internal ribose entry elements called IREs and Iron response stem loop structures, also called IREs.
The 3’UTR contain many sequence elements such as Antisense RNA binding sequences, regulatory stem loops like IRE-BP, ARE-AUUUA sequences, mRNA localization sequences called Zip Code, EDEN-embryonic deadenylation elements, DICE-Differential control elements, CPE-cytoplasmic adenylation elements, mi/siRNA binding elements, SECIS element-selenocysteine binding sequence, CRE-cysteine rich stability element, CITE-3’cap independent translation enhancer element, and SL- stem loop element.
Structural organization of eukaryotic mRNA and the different points of possible regulation of translation through various trans-acting factors;5′-m7G, cap structure; eIF, eukaryotic initiation factor; CPE, cytoplasm polyadenylation element; EDEN, embryonic deadenylation signal; DICE, differential control element; PABP, poly(A)-binding protein; the possible sites of interaction of transacting factors (yet many unknown) in the coding sequence. Regions of mRNA involved in subcellular localization and stability are also indicated. ; Sangeeta Chaterjee and Jayant K.Pal; http://www.biolcell.org
Baker and Coller; http://genomebiology.com
Regulation of eukaryotic mRNA translation occurs at numerous control points. Recognition of 3' UTR sequence or structural elements (green and red boxes) by RNA-binding proteins leads to either activation or repression of translation, often through alteration of the 3' poly(A) tail or through interactions with proteins that bind at the 5' terminal cap structure (that is, the initiation factor eIF4E or cap-binding proteins). Repression of translation by miRNAs can occur through inhibition of translation initiation or elongation, and may also lead to changes in the status of the mRNA 3' poly-(A) tail. Elements found within the mRNA 5' UTR (yellow box) can bind regulatory proteins that repress translation by inhibiting 48S ribosome scanning. Global regulation of mRNA translation is commonly achieved through modification of the translational apparatus (that is, by phosphorylation of the translation initiation factors eIF2α and eIF4E) and the ribosome itself, or modulation of protein partner binding affinities (such as the phosphorylation of the eIF4E-binding proteins). Translation can be initiated independent of the mRNA 5' cap through a structured internal ribosome entry site (IRES) in the 5' UTR whose efficiency in initiating translation is, in turn, modulated by trans-acting factors (ITAFs).
RNA structure: new messages in translation, replication and disease; mRNA with many 5’ and 3’ regulatory elements- %’ UTR may contain pseudo knot, IREs and 3’ may contain Zip code, CBE,CITE, mi antisense sequence. ,
Schematic illustration of discreet RNA regulatory elements: The translation of an mRNA is regulated by diverse mechanisms that involve both structural and non-structural RNA elements, as well as interactions with RNA-binding proteins. IREs found in the 5' UTR promote cap-independent translation. Pseudoknots can be located in the 5' UTR, the 3' UTR, or the coding region, and their localization influences their effect on translation; for example, initiation, frameshifting and termination. 3' UTR structural elements such as C rich stability enhancer elements CREs, 3’Cap independent translational enhancer element-CITEs and Stem loop SLs often function through long-range RNA interactions. The miRNA target sites are located in the 3' UTR. The canonical mRNA features of the Cap (m7G) and poly-(A) are targets of several regulatory interactions. The RNA-binding protein-binding sites are shown as blue ovals, and the miRNA-binding site is shown as a brown rectangle. AUG, initiation codon; CITE, cap-independent translational enhancer; IRES, internal ribosome entry site; m7G, 7-methyl-guanosine; miRNA, microRNA; mRNA, messenger RNA; ORF, open-reading frame; SL, stem loop; UTR, untranslated region.
The generic structure of a eukaryotic mRNA, illustrating some post-transcriptional regulatory elements that affect gene expression. Abbreviations (from 5' to 3'): UTR, untranslated region; m7G, 7-methyl-guanosine cap; hairpin, hairpin-like secondary structures; uORF, upstream open reading frame; IRES (iron response), internal ribosome entry site; CPE, cytoplasmic polyadenylation element; Zip lock, antisense elements, Iron response elements 3’IRES, ARE elements, AAUAAA, polyadenylation signal.; Mignone er al;http://genomebiology.com/; http://openi.nlm.nih.gov/
Types of mRNA zip codes; mRNA localization; message on the move; Ralf-Peter Jansen; http://www.nature.com/
a | The Vg1 3' UTR contains a 340-nucleotide zip code that is sufficient for mRNA localization, termed the Vg1LE, or Vg1 localization element. The functional units in this type of element are short repetitive sequences (either UAUUUCUAC or UUCAC)59, 104. b | The bicoid localization element (BLE) in the bicoid 3' UTR is an example of a zip code with a modular architecture. bicoid mRNA undergoes several sequential transport steps, each involving different, partially overlapping, regions in the highly structured 3' UTR. (Light blue, early localization; dark blue, early and late localization; purple, mRNA anchoring.) c | ASH1 mRNA is an example of a zip code element that lies in the coding region (E1, E2a, E2b) and in the 3' UTR (E3). The E3 element also represents an example of a structure-based zip code, because the displayed stem loop structure but not the primary sequence is important for the function of the zip code
Firoz Ahmed et al; http://journal.frontiersin.org/
. UTR, untranslated region; CDS, coding sequence; m7G, 7-methyl-guanosine cap; IRE, iron-responsive element; uORF, upstream open reading frame; IRES, internal ribosome entry site; ARE, AU-rich element; PAS, poly(A) signal, 1, et al
Even tissues, which are kept in dark and etiolated, when, exposed to light or hormones or both, mRNAs are activated and then they are translated. Such mRNA under conditions such as mentioned above or cells remain in stationary phase dictated by cell cycle events loose most of the poly-A tail. Such dormant mRNAs at 3’ UTR contain CPE cytoplasmic poly adenylation elements bound by CPEB which in turn bound by Maskin proteins. The Maskin in turn binds to TF4E thus the mRNAs are in circular state and rendered inactive under dark or unfavorable conditions.
Luc Paillard H. Beverley Osborne
Conserved PUF mechanisms of mRNA control. A: FBF binding elements (FBEs) were among the first PUF regulatory elements identified; most are located in the mRNA's 3' untranslated region (3'UTR). B: PUF (Pumilo and FBF (fem-3 binding factor) proteins recruit deadenylase to repress target mRNAs. C: Both nematode FBF and human PUM2 form a ternary complex with an Argonaute (Ago) and translation elongation factor (eEF1A) to repress translation; Judith kimble; http://www.hhmi.org/
Translational regulation of the transferrin receptor and ferritin production; Production of the transferrin receptor (TfR) and ferritin is regulated at the level of mRNA by iron regulatory proteins (IRPs), which bind to iron response elements (IREs) on the 3'- and 5'- untranslated regions of their respective mRNAs1. a | In iron deficiency, the IRPs bind to the IREs, protecting the TfR mRNA from nuclease digestion and preventing the synthesis of ferritin. b | When iron is abundant, the modified IRP no longer binds to the IREs — in IRP1 the IRE binding site is blocked by a 4Fe–4S cluster (green rectangle), whereas in IRP2 the protein is targeted for destruction in the proteasome — allowing TfR mRNA to be destroyed and allowing the expression of ferritin; Luigi Zecca, Moussa B. H. et al; Nature Reviews; http://www.nature.com/
Some of these mRNA also contain EDEN (Embryonic Deadenylation Element), which are bound by EDEN binding proteins. EDEN-BP binding sequence is discerned as UGUCCUUUUAUAUGUAA or UR repeats and a single 36 ntd region -UAUAUGUAUGUGUUGUUUUAUGUGUGUGUGUGUGCU.
Evolutionary conservation of deadenylation by CELF1 protein and GU-rich sequences. (a). In and eggs, after fertilization, EDEN-BP (CELF1 homologue) bound to EDEN-containing maternal mRNAs, causing deadenylation and subsequent translational activation.(b). In mammalian cells, CELF1 binds to GREs within the 3' UTR of specific transcripts and promotes their deadenylation (by deadenylases) and subsequent decay by the Exosome; ( (CUGBP and embryonically lethal abnormal vision-type RNA binding protein 3-like factor 1 ); Daniel Beisang et al; http://www.intechopen.com/
Binding of CPEB to UUUUAU sequence and regulating cytoplasmic polyadenylation @2001 Nature Publishing group, Mendez,R and Richeter, J.D, Nature Review Molecular Biology, The fertilized eggs contain all the required mRNAs for rapid cell division and also contain factors to prevent premature translation and remain dormant; Maternal mRNA and PolyA Tail in Oocytes; Ren-Jang Lin, Ph.D; http://www.nature.com/
Model for CPEB activity. Some mRNAs contain a cytoplasmic polyadenylation element (CPE), which is bound by CPEB. CPEB also interacts with Maskin (or Neuroguidin, a functionally related protein), which in turn interacts with the cap (m7G) binding protein eIF4E. Such mRNAs are translationally inactive because Maskin inhibits the association of eIF4E and eIF4G, another initiation factor that helps recruit the 40S ribosomal subunit to the 5′ end of the mRNA. Cues such as NMDA receptor activation stimulate the kinase Aurora A, which phosphorylates CPEB, an event that causes poly(A) tail elongation. poly(A) binding protein (PABP) binds the newly elongated poly(A) tail and recruits eIF4G. The PABP-eIF4G dimer helps to displace Maskin from eIF4E, allowing eIF4G to bind eIF4E and initiate translation. R. Suzanne Zukin et al ;http://journal.frontiersin.org/
Model of polyadenylation-induced translation. Dormant CPE-containing mRNAs (e.g. cyclin B1) in immature oocytes are bound by CPEB, which in turn is bound to maskin, which in turn is bound to eIF4E, the cap-binding factor. The binding of maskin to eIF4E precludes the binding of eIF4G to eIF4E, thus inhibiting the formation of the initiation complex. The cleavage and polyadenylation specificity factor (CPSF) may or may not be loosely associated with the hexanucleotide AAUAAA at this time. Following progesterone stimulation, the kinase aurora is activated and phosphorylates CPEB Ser174, an event that causes CPEB to bind and recruit CPSF into an active cytoplasmic polyadenylation complex, presumably helping it to associate with the AAUAAA. CPSF recruits poly(A) polymerase (PAP) to the end of the mRNA, where it catalyzes poly(A) addition. The newly elongated poly(A) tail is then bound by poly(A)-binding protein (PABP), which in turn associates with eIF4G. eIF4G, when associated with PABP, then displaces maskin from, and binds to, eIF4E, thereby initiating translation. eIF4G, through eIF3, interacts with the 40S ribosomal subunit. Model of polyadenylation-induced translation.
R. Suzanne Zukin et al;‘http://journal.frontiersin.org/
Model of polyadenylation‐induced translation. Dormant CPE‐containing mRNAs (e.g. cyclin B1) in immature oocytes are bound by CPEB, which in turn is bound to maskin, which in turn is bound to eIF4E, the cap‐binding factor. The binding of maskin to eIF4E precludes the binding of eIF4G to eIF4E, thus inhibiting the formation of the initiation complex. The cleavage and polyadenylation specificity factor (CPSF) may or may not be loosely associated with the hexanucleotide AAUAAA at this time. Following progesterone stimulation, the kinase aurora is activated and phosphorylates CPEB Ser174, an event that causes CPEB to bind and recruit CPSF into an active cytoplasmic polyadenylation complex, presumably helping it to associate with the AAUAAA. CPSF recruits poly(A) polymerase (PAP) to the end of the mRNA, where it catalyzes poly(A) addition. The newly elongated poly(A) tail is then bound by poly(A)‐binding protein (PABP), which in turn associates with eIF4G. eIF4G, when associated with PABP, then displaces maskin from, and binds to, eIF4E, thereby initiating translation. eIF4G, through eIF3, interacts with the 40S ribosomal subunit; http://emboj.embopress.org/
Model for CPEB activity: Some mRNAs contain a cytoplasmic polyadenylation element (CPE), which is bound by CPEB. CPEB also interacts with Maskin (or Neuroguidin, a functionally related protein), which in turn interacts with the cap (m7G) binding protein eIF4E. Such mRNAs are translationally inactive because Maskin inhibits the association of eIF4E and eIF4G, another initiation factor that helps recruit the 40S ribosomal subunit to the 5′ end of the mRNA. Cues such as NMDA receptor activation stimulate the kinase Aurora A, which phosphorylates CPEB, an event that causes poly(A) tail elongation. poly(A) binding protein (PABP) binds the newly elongated poly(A) tail and recruits eIF4G. The PABP-eIF4G dimer helps to displace Maskin from eIF4E, allowing eIF4G to bind eIF4E and initiate translation.
Most of the hnRNPs 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 hnRNPs proteins (not all):
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.