Capping and Poly-Adenylation:

 

 

The nascent pre mRNA is long contains various elements which are to be cut and processed; and it also undergoes various modifications fit to be translated into a functional protein.

 

 

Pre mRNA goes through various processing to generate functional translatable RNA

 

Capping and its importance:

 

 

Capping complex bound to CTD tail

 

Phosphorylated RNAPII CTD tail associated capping complex performs capping at the 5’ end of the pre-mRNA. The CTD tail has the repeats of seven amino acids-TSPYTSPS; (Threo--Ser-Pro-Tyr-Threo-Ser-Pro-Ser); the S. Y and T are the sites for phosphorylation. This sequence is repeated 22 to 52 times in many such RNAPII enzymes.

 

 

 

This illustration shows the process of capping starts on P-lated CTD tail as the 5’end of the mRNA emerges from the RNAPII complex; the required components are already assembled on the CTD tail.

 

 

 

As the 5’ end of the pre mRNA emerges out of RNAP II complex, the CTD bound capping complexes add Guanine (GP) to the first pA or pG (N) of the precursor mRNA. Eukaryotes have designed to have multisubunit complex associated with CTD complex tail. The complexes perform Capping, Splicing and poly-A addition as the mRNAs are transcribed.

 

 

 

 

 

 

7’CH3 G-5’P-P-P-5’A---- = Cap-0

2’CH3 A-- =cap1

 

This diagram shows structural elements of cap structure.

 

 

7’CH3.G5’p.p.p+p.p.p5’A.n.n-à 7’CH3.G.5’ap.pb.pa.5’ A.n.n—

 

 

 

7-CH3G Mono methylated Cap

 

2’2’7CH3G Trimethylated Cap

 

ARCA Anti reverse cap analogue.

 

In Orthomyxovirus viruses, addition of cap like structure to its RNA or (+) RNA is entirely different.  Viral enzymes choose host’s capped mRNAs and cut the leader sequence with few more nucleotides at A or G, then adds this segment to its 5’ end of viral (+)-sense RNA; a process called “capping by molecular stealing”.  In eukaryotes Cap structure is not just the part of pre-mRNAs; but cap structure is added many small Mol.wt. Sn RNAs though they are non-coding RNAs.  Some viral RNAs use 5’VpG as the cap. Capping takes place very early during mRNA synthesis.

Cap Analogs –Enhance mRNA stability and translation efficiency (Jeno Bioscience):

Many eukaryotic and viral mRNAs are modified at their 5' ends by addition of 7-Methylguanosine (N7-methyl guanosine or m7G), known as "Cap". "Capping" of the mRNA structure plays a crucial role in a variety of cellular processes which include translation initiation, splicing, intracellular transport and turnover.  Capped mRNAs are generally more efficiently translated in wheat germ and reticulocyte in vitro translation systems, and they are less susceptible to exonuclease degradation during microinjection experiments compared to uncapped mRNAs.  However it is to be noted that the unmethylated cap analog shows no significant difference in its translation properties compared to the monomethylated cap analog

 

In Ascaris lumbricoides transcription of the spliced leader RNA was catalyzed by RNA polymerase II, and the majority of the spliced leader RNAs synthesized in vitro possessed a trimethyl guanosine cap structure identical to that found on in vivo-synthesized spliced leader RNA.

 

It should e noted capping of mRNA can be RNA Cap Analog: 1. Unmethylated G, 2. G[5']ppp[5']G, 3. Monomethylated, m7G[5']ppp[5']G.  Capping of mRNA can be done in an in vitro conditions too.

 

Primitive eukaryotes like Caenorhabditis elegans produce mRNAs capped with either m7GTP or m32,2,7GTP. Caenorhabditis elegans also expresses five isoforms of the cap-binding protein eIF4E. Some isoforms (e.g. IFE-3) bind to m7GTP–Sepharose exclusively, whereas others (e.g. IFE-5) bind to both m7GTP- and m3 2, 2, 7 GTP–Sepharose; Hiroshi Miyoshi2, Donard S. Dwyer3, Brett D. Keiper1, Marzena Jankowska-Anyszka4, Edward Darzynkiewicz, EMBO (2002).

 

Importance:

 

Pre mRNA Transcription Termination Site and Processing 1:

 

The 3’ end of the mRNA genes has many regulatory elements which perform functions like stability of mRNA, elements for degradation of mRNA, transport to certain loci in the cell, elements that make mRNA as informosomes remain untranslated, making mRNA inactive for the storage; also has site for the binding of mi/siRNA for inactivating or degradation of mRNA and poly-A addition elements.  But transcription of mRNA is terminated beyond these elements.  The termination site of transcription varies from one gene to the other.  The distance from poly-A site and termination site varies.  Most of the pre mRNAs are cut at the 3’ region at specific site and poly-As are added.  In some such as histone, 3’ processing is different for they don’t contain poly A tail but one of the Histone mRNA is added with Poly(A) tail, very unusual.

 

Eukaryote transcription termination distance from Poly-A site

 

Gene

Distance from poly-A signal site

D-globin

600bp

A-globin

1500bp

Globin-mice

100-300

Globin-human

100-300

DHFR-mice

1000-2000

a-Amylase

2000-4000

Ovalbumin

800-1000

Gastirin

192

 

 

 

 Addition of poly- (A) Tail and Its Importance:

 

 

CPSF:  Cleavage and poly adenylation specific factor is a complex of four subunits such as 160kd, 100kd, 70kd and 30 KD each.  The complex binds to AAUAAA sequence, by doing so it specifies a nearby position of poly-A (adenylation) start site.  This complex also interacts with Poly-A polymerase and cleavage stimulation factor (CStF).

 

CStF: Cleavage stimulation factor identifies and binds to GU n U rich site.  This identifies the site for cleavage.  This complex consists of 77, 64, 50 KD protein subunits.  This interacts with cleavage factors such as CF-1 and CF 2.

 

CF1 and CF-2:  Cleavage factors CF1 and CF2 associate with CPSF and CStF.  The enzyme consists of 68, 59 and 29 KD subunits.  These are cleavage enzymes.

 

PAP:  The molecular mass of Poly-A polymerase is 82kd, a monomer.  It is required for facilitating cleavage and catalyzing poly-A addition.  For its activity, it also requires poly-A binding protein-II (PAB-II).  The enzyme is 689 aa long and contains, N-terminal, RBD domain (RNA binding domain), PM domain (polymerase domain), and NLS1 and NLS2 sites for nuclear localization (?).  They also have serine and threonine rich sites for phosphorylation.

 

PAB-II:  This33 KD protein, is the counter-part of cytoplasmic poly-A binding factor (PAP-I).  The binding of this factor stimulate PAPs activity and also it regulates the length of poly-A tail and provides safety from 3’exonucleases.

 

 

This diagram shows the process of Polyadenylation.

 

yeast polyadenylation complex

 

Poly adenylation in yeast cells: Yeast lacks a distinct AAUAAA sequence, and polyadenylation is associated with less well defined A/U rich and A rich sites. The CFPS equivalents are Cft1, Cft2 and Yth1. A factor comparable to CstF, Rna15/Rna14/Pfs2, binds upstream of CPFS a number of factors appear to be involved in binding downstream of the cleavage site as for mammalian CstF. Pap 1p is the polyA polymerase. Two-hybrid screens have shown it to interact with protein Fip 1p which in turn bridges to the CstF-like factor Rna14/Rna15, which then binds to a protein complex associated with an additional upstream A/U rich sequence marker. An hnRNP-like protein Hrp1 binds in between the CstF and CPSF equivalent factors.

 

 

Cleavage factors PfsIA (CstF), IB are involved in cleavage, but functions do not correspond exactly to the mammalian counterparts.

 

 

 

This diagram illustrates different steps in the addition of Poly(A) tail.

 

 

 

Mechanism:

 

 

 

Pre mRNA Transcription Termination Site and Processing 2:

 

Processing of Histone precursor mRNAs:

 

Transcriptional processing of histone transcripts requires specific factors and specific small molecular weight RNAs.  Histone pre mRNA contain a cap at the 5’end and no poly (A) tail.  Histone transcription is replication dependent.  Histone mRNA is stored in frog oocytes and its translation is activated with fertilization. As the transcription of histone mRNA proceeds towards the end region of the gene, complementary base pair sequences generate a small stem loop (SL) structures followed by a specific sequence of nucleotides.  However in the oocytes of Xenopus short oligo-As are added (by PRAPP) to histone mRNA at the end of stem loops. The presence of oligo-As and SLBP (31kDa) binding proteins (SLBP1 and SLBP2) prevent initiation of translation. Te stored mRNAs in oocytes is bound by FRGY2 translation repressor proteins.  SLBP2 is cytoplasmic and binds to histone mRNA when histone mRNAs are stored and remain inactive. It has RNA binding and RNA processing domains.  However at meiosis the poly-A oligo’s are removed. It is how Xenopus histone mRNA is regulated in oocytes. Histone mRNAs of protozoa, plants, and yeast are polyadenylated (Chaubet et al. 1988). Interestingly histone mRNAs does not contain introns. In drosophila histone mRNA are stored in oocytes required for development.  Even in frogs histone mRNAs are stored with SLBP2 bound.  During early embryogenesis histone mRNA transcribed heavily for the development requirement.  SLBP1 is required for histone translation activity.  SLBP2 inhibits translation.

The U7 snRNA also contains a small stem loop and specific sequences at its 3’end, which are complementary to the terminal region of histone pre-mRNA transcript. 

The U7 Sn RNA with its secondary structure base pairs with the 3’ region ACCCA of the transcript. 

·         As transcription proceeds beyond this region, a nuclear factor binds to 3’ end of the transcript and stops transcription and terminates the process. 

 

The nuclear factor that contains a heat labile protein component binds to the stem loop structure of the transcript just upstream of the cleavage site. Transcriptional termination of histone transcripts requires specific factors and specific small molecular weight RNAs.  As the transcription moves to the end region of the gene, complementary base sequences generate stem loop structures followed by a specific sequence of nucleotides.

 

Another factor U7 SnRNA/snRNPs which is 56 ntd long also contains a stem loop structure at its 3’ end and a 7’methylated Cap at the 5’end. It also contains sequences at its 5’end, before its stem loop, which are complementary to the terminal region called HDE (Histone Downstream Elements) of histone pre-mRNA transcript. 

 

 

 

 

 

 

 

 

 

The U7snRNA contains secondary structure and it is bound by SMN heptapeptide proteins.  The snRNA base pairs with the ‘UGGGU’ sequence of the mRNA transcript at 3’ end next to stem loop region.  This leads to the association of stem loop binding proteins SLBPs and CPSF and CsTFs to this stem loop structure. The bound factors cut the 3’ end of histone mRNA

 

 

This pairing leads to the cleaving of the transcript 4-5 nucleotides downstream of the stem loop at CACCCA found in the mRNA.

 

Full-size image (7 K)

Possible alignment of structural elements of the U7 snRNA and the histone pre-mRNA resulting from formation of a duplex between the U7 snRNA 5′ end and the histone downstream element (HDE). Sequences of the mouse H2a-614 pre-mRNA near the processing site and mouse U7 snRNA are shown. The possible Watson–Crick base pairs (vertical lines) and GU base pairs (dots) between the 5′ end of U7 snRNA and the HDE are indicated. Zbigniew Dominski, William F Marzluff.

Representation of the processing reaction occurring at the 3′ end of replication-dependent histone precursor mRNA;. The endonucleolytic cleavage (indicated by the scissors) occurs between the highly conserved hairpin (see also Fig. 3A) and the purine-rich sequence HDE. Trans-acting factors are hairpin binding protein (HBP), the U7 snRNP and zinc finger protein 100 (ZFP of 100 kDa).

Histone pre-mRNA processing: Processing is initiated by binding the SLBP to the stem-loop and by binding the U7 snRNP to the HDE, which makes base-pairing contacts with the 5′ end of U7 snRNA. Binding of the U7 snRNP to the pre-mRNA is stabilized by interactions between a U7 snRNP protein, hZFP100 and SLBP. The two novel Sm proteins, Lsm10 and Lsm11, are indicated by the red and green balls. Following cleavage of the pre-mRNA, which probably requires factors in addition to U7 snRNP, SLBP remains associated with the mature histone mRNA; Zbigniew DominskiWilliam F Marzluff

 

Recently, Utz.Fischer and his team has shown that the formation of spliceosomal U7-snRNPs requires assisting factors including the Survival Motor Neuron (SMN) proteins, mentioned earlier, the gene product that affects the neuromuscular and spinal muscular atrophy. SMN is part of a multiprotein (SMN-) complex that facilitates cytoplasmic assembly of U snRNPs in vivo in an ATP-dependent reaction. In this process, spliceosomal Sm proteins, which constitute the protein core of U snRNPs, must bind to the SMN-complex prior to their transfer onto the U snRNAs. The SMN-complex itself is functionally regulated by a methyltransferase, termed PRMT5-complex. This complex catalyzes the symmetric dimethlyation of arginines in some Sm proteins, thereby increasing their affinity for the SMN-complex.  Among the seven Sm proteins 5 are common to all and two are specific to U7 snRNAs, they are smlike called LSM10 and Lsm11 substitute for SMN proteins D1 and D2.

More recent studies indicated that not only the assembly of spliceosomal U snRNPs but also the histone-mRNA processing U7 snRNP particle requires assisting factors. Specifically, it is now known that a minor SMN-complex containing U7-specific factor mediates the formation of the U7-particle.

 

 

 

 

 

Symplekin and multiple other polyadenylation factors participate in 3’end maturation of histone mRNAs: Kolev NG, Steitz JA.

 

Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3' ends. Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoproteins (U7 snRNP) and an unidentified heat-labile factor (HLF). The above authors describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and Symplekin. Reconstitution experiments reveal that Symplekin, previously shown to be necessary for cytoplasmic poly(A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation.

 

A 3' exonuclease that specifically interacts with the 3' end of histone mRNA:

Dominski Z, Yang XC, Kaygun H, Dadlez M, Marzuluff WF.

 

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein that binds to this region. Using RNA affinity purification, we identified a second protein, designated 3’hExo that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/ or inhibition of DNA replication.

 

The polyadenylation factor CPSF-73 is involved in histone-pre-mRNA processing: Dominski Z, Yang XC, Marzluff WF;Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.

 

During 3' end processing, histone pre-mRNAs are cleaved 5 nucleotides after a conserved stem loop by an endonuclease dependent on the U7 small nuclear ribonucleoproteins (snRNP). The upstream cleavage product corresponds to the mature histone mRNA, while the downstream product is degraded by a 5'-3' exonuclease, also dependent on the U7 snRNP. To identify the two nuclease activities, we carried out UV-cross linking studies using both the complete RNA substrate and the downstream cleavage product, each containing a single radioactive phosphate and a phosphorothiolate modification at the cleavage site. We detected a protein migrating at 85 kDa that cross linked to each substrate in a U7-dependent manner. Immunoprecipitation experiments identified this protein as CPSF-73, a known component of the cleavage/polyadenylation machinery. These studies suggest that CPSF-73 is both the endonuclease and 5'-3' exonuclease in histone-pre-mRNA processing and reveal an evolutionary link between 3' end formation of histone mRNAs and polyadenylated mRNAs.

 

Molecular mechanism of histone pre-mRNA 3’ end processing by U7 snRNP. Symplekin and multiple other polyadenylation factors participate in 3’end maturation of histone mRNAs: Kolev NG, Steitz JA.

 

 

U7

U7 RNA

Most metazoan messenger RNAs encoding histones are cleaved, but not polyadenylated at their 3' ends. Processing in mammalian cell extracts requires the U7 small nuclear ribonucleoproteins (U7 snRNP) and an unidentified heat-labile factor (HLF). We describe the identification of a heat-sensitive protein complex whose integrity is required for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor (CPSF), two subunits of the cleavage stimulation factor (CstF), and Symplekin. Reconstitution experiments reveal that Symplekin, previously shown to be necessary for cytoplasmic poly (A) tail elongation and translational activation of mRNAs during Xenopus oocyte maturation, is the essential heat-labile component. Thus, a common molecular machinery contributes to the nuclear maturation of mRNAs both lacking and possessing poly(A), as well as to cytoplasmic poly(A) tail elongation.

 

A 3' exonuclease that specifically interacts with the 3' end of histone mRNA: Dominski Z, Yang XC, Kaygun H, Dadlez M, Marzuluff WF.

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/ or inhibition of DNA replication.

At the end of translation SLBp proteins are phosphorylated and released.  The released SLB proteins are ubiquitylated and degraded. The stem-loop of histone mRNA recognized by 3’exonucleases and the same are degraded.

 

Post-transcriptional control of animal histone gene expression—not so different after allPamela Nicholson and Berndt Müller

 

graphical abstract image (ID: b802133c)

Histone proteins are essential components of eukaryotic chromosomes. The expression of histone genes is cell cycle controlled and coupled to DNA replication, to ensure the packaging of replicated DNA into chromatin. The post-transcriptional control of histone gene expression is a key element in this coupling to DNA replication. It involves mRNA 3 end formation by histone-specific nuclear RNA processing, which produces mRNAs lacking a poly (A) tail, translation and mRNA stability control. This requires several histone-specific trans-acting factors and was thought to be a special case. Here we review recent observations that now reveal that many of the factors involved are shared with processing, translation and degradation of poly (A) mRNA

 

Poly A-containing histone H4 mRNA variant (H4-v.1):

Isolation and sequence determination from bovine adrenal medulla:

Nathalie Gendron, Michel Dumont, Marie-France Gagné and Simon Lemaire*

 

A histone H4 cDNA variant (H4-v.1) was cloned from a bovine adrenal medullary phage library using PCR as a method of detection. The isolated clones contained a short 5′ untranslated region (UTR) followed by the histone H4 coding region and a long atypical 3′UTR. The 3′UTR comprised the palindromic and purine-rich sequences typical of cell-cycle dependent histone mRNAs, and a 1.1 kb extension downstream of the palindromic sequence ending with a poly (A) track typical of cell-cycle independent histone mRNAs. Northern blot and RT-PCR analyses indicate that the transcript is fully expressed in bovine adrenal medulla. Thus, bovine histone H4-v.1 mRNA represents the first example of a histone H4 transcript that contains both 3′UTR characteristics of cell-cycle dependent and cell-cycle independent histone mRNAs.

 

 

 

 Object name is mb1231933009.jpg

 

Fig:  Negative coupling of processing to transcription. The model explains how processing might be inhibited by RNA polymerase II arrested downstream of the processing site. (A) RNA polymerase II paused at the arrest site leaves just enough RNA exposed to allow binding of SLBP and the U7 snRNP and processing of the transcript. (B) When the polymerase backslides and enters the arrested conformation, the HDE (black box) is no longer accessible to the U7 snRNP, and processing is inhibited. (C) Comparison of the 3′ regions of H4, H3, and H2b. Starting with the last two nucleotides of the stem-loop RNA, sequences from H4, H3, and H2b were aligned. Underlined text, purine-rich region; boldface text, T-rich region; boldface underlined text, pause-arrest site

 

 

 

Functions of Poly (A) tail: