1. Introduction
1.1. A multitude of RNAs
The central dogma of molecular biology stated that genetic information only flows in one direction, from DNA to proteins via an intermediate called messenger ribonucleic acid (mRNA) [1,2,3]. Originally, ribonucleic acid (RNA) was thought to have roles in information transfer and structure maintenance. Today, we know that RNA performs a remarkable range of functions in the living cell, (control of gene expression, chromosome –end maintenance, housekeeping activities, sorting of proteins in the cell and defines metazoan development) [3].Although, proteins have enzymatic activities mostly, in the early 1980s has been shown that RNA molecules can catalyze a chemical reaction and RNAs with catalytic activity are called
Small nuclear RNAs are components of the macromolecular machinery (spliceosome) that has a role in the maturation of mRNA. They are termed
1.2. Structure of RNA and association with proteins
DNA and RNA have similar covalent structures, the only difference being the change from a 2`-deoxyribose sugar to a ribose sugar and from a methyl group in thymine to a hydrogen in uracil. RNA has much wider biological activities and adopts a wider range of structures. DNA double helices preferentially assume the B-form structure in solution and RNA double helices are found in the A-form. The
1.3. All mRNA processing steps are coupled
Eukaryotic gene expression is a complex, stepwise process that begins with transcription (synthesis of pre-mRNA) [18]. Mature mRNAs are produced in the cell nucleus from primary transcripts of coding genes (pre-mRNAs) by a series of processing events which include capping, splicing, and 3` end polyadenylation. Mature mRNAs are transported to the cytoplasm. All modification steps are coupled and influence each other. RNA polymerase II is a key molecular coordinator of these processing events, and phosphorylation of it has regulatory role [19, 20,21,].
1.4. Removal of introns and the splicing reaction
In 1977, a number of research groups discovered that the genes of higher organisms are often made up of a sequence of coding (called exons) and non-coding base sequences (introns). During transcription, all parts of the gene are copied to form a strand of pre-mRNA. Introns are removed and the exons stitched together so that the now continuous exons can be translated to produce a protein. This splicing of the pre-mRNA is a multistage process, carried out by complex macromolecular machinery known as the spliceosome, which is among the most complex macromolecular machineries in the cell [22].
Splicing of precursors to mRNAs occurs in two steps, both involving a single transesterification reaction [23].Assembly and function of the spliceosome requires approximately 300 polypeptides and five snRNAs, not considering gene-specific RNA-binding factors [23]. There are two distinct types of spliceosome in most cells. The major class U2-type spliceosome is universal in eukaryotes, whereas the minor class or U12-type spliceosome is not present in some organisms. The evolutionary relation between these two spliceosomes is uncertain.
1.5. Types of introns
The pre-mRNA contains conserved elements at its intron/ exon boundaries that determine the proper sites for the splicing reaction (Figure 3.). The 5’ splice site contains a conserved consensus sequence, which is AG/GURAGU (R=purine, / denotes the exon/ intron boundary). The branch site lies between 100 and 18 bases upstream of the 3’ splice site and has the consensus: CUR
1.6. Small nuclear ribonucleoproteins, snRNPs
Small ribonucleoproteins (RNPs) are tight complexes of one or more proteins with a short RNA molecule (usually 60-300 nucleotides). RNPs inhabit nuclear and cytoplasmatical compartments of the eukaryotic cell [25]. Those that reside in the nucleus, the small nuclear ribonucleoproteins (snRNPs) can themselves be divided into two families. There are
1.7. U snRNP biogenesis
Subsequent to transcription by RNA polymerase II and capping, pre-U1 snRNA assembles with several factors including cap-binding proteins (CBP), a phosphorylated adaptor for RNA export (PHAX), Crm1, and Ran-GTP, which all together mediate export of U1 snRNA to the cytoplasm. After export, Sm proteins interact with the U snRNAs to form the snRNP Sm core. This step is facilitated by the SMN complex (survival of motor neurons complex). The SMN complex is composed from SMN protein and the other proteins called Gemins (Gemins 2-7). Nuclear re-import is mediated by snurportin-1 (SPN1), which binds to the snRNAs m3G cap structure. After import, these factors dissociate. The U1 specific proteins are imported independently into the nucleus, where assembly into mature U1 snRNP occurs [27]. This is a pathway shared with U1, U2, U4 and U5 snRNPs.
1.8. Spliceosome assembly
Assembly of a spliceosome for excision of an intron requires recognition of sequences at the 5' splice site as well as the branch site and nearby 3' splice site. U1 snRNA binds to the 5` end of the intron using sequence complementarities. There are reports which show that the U1 snRNA recognizes the 5` splice site in a preassembled penta-snRNP complex [28]. U2 snRNP complex associates with the branch region. Early snRNP/pre-mRNA complexes are preferentially committed to splicing as compared to free RNA and thus are called
The classical view of spliceosome assembly has been challenged by Stevens et al [30]. This group isolated from yeast a penta-snRNP complex which when supplied with soluble components, does splice pre-mRNA.
1.9. mRNA stabilization, degradation
Regulation of mRNA decay rates is an important control point in determining the abundance of cellular transcripts. Some mRNA has half-lives that are 100 times shorter than cellular generation times and some mRNA have half-lives spanning several cell cycles [21]. The poly (A) tail is important in stabilization of mRNA. It interacts with the poly (A) binding protein (PABP), which makes direct contact with a specific region of the translation-initiation factor (eIF4E). Translation initiation factor (eIF4) interacts with the cap binding proteins. In this way, a ternary (PABP-translation initiation,-cap binding protein, poly (A) tail) complex is formed which circularizes mRNA
2. Family of Sm-LSm proteins
2.1. Sm proteins, assembly of U1, U2, U4, U5 snRNPs
The Sm proteins were first discovered as antigens targeted by so-called
Solved structures of this protein family members (pdb codes: 1d3b,1b34,1hk9, 1h64,1i8f,1i4k,1kq1,3bw1,1th7) show that the fold is highly conserved. It is defined by an N-terminal helix, followed by a five-stranded anti-parallel β sheet. Strands β1, β2, and β3 are part of the Sm1 motif, whereas the Sm2 motif forms strands β4 and β5. The five stranded β sheet is strongly bent in the middle and the conserved hydrophobic residues form a hydrophobic core [41].
The Sm proteins bind to the Sm site of U snRNAs [42]. The Sm site consensus sequence (PuAU4-6GPu) has a central, uridine rich tract and flanking purines.
2.2. LSm proteins
Sm and Sm-like proteins are found in all kingdoms of life:
Similar to canonical Sm proteins, the LSm proteins are recognized by antibodies from patients suffering from systemic lupus erythematosus (SLE) [59]. Sm/LSm proteins always appear as homomeric (in the case of prokaryotes) or heteromeric (in eukaryotes) ringlike multimers. These ring-shaped complexes, generally containing either six or seven subunits, are the functional LSm protein unit. All canonical Sm proteins are essential for vegetative growth of yeast. LSm proteins have variable effects after depletion in yeast. In mice embryos, LSm4-null zygotes survived to the blastocyst stage, but died shortly after [60].
2.3. Role of LSm 2-8 oligomers in U6snRNP assembly
The LSm2-8 complex was isolated from Hela cells nuclear extract in an RNA free form. Electron micrographs revealed a doughnut–shaped heterooligomer, similar to the Sm core snRNPs [58]. LSm proteins have a high affinity for single-stranded oligo-U, but they do not recognize the canonical Sm binding site. In yeast and humans, LSm2-8 forms a heteroheptameric ring around the 3` end of U6 snRNA, consisting of a U rich tract. The Sm core RNP is extremely salt stable; however, LSm-U6 snRNA dissociates at salt concentrations higher than 0.5M, or in the presence of competitor RNA, suggesting that the LSm-U6 complex is less stable [58]. U6 snRNA has no conserved Sm site and does not associate with Sm proteins. Its biogenesis pathway differs in many respects from the U1, U2, U4 and U5snRNP pathways; it is transcribed by RNA polymerase III and capped by γ-monomethyltriphosphate. The 3` end of pre-U6 snRNA is elongated during maturation and subsequently trimmed leaving in most organisms a 2`-3` cyclic phosphate. The enzymes involved in this process are specific for U6 snRNA, and U6 snRNA does not leave the nucleus [61]. Mature U6 snRNA shows nucleoplasmic localization [62]. Experimental evidence suggests that U6 snRNA is present in the cytoplasmic compartment of mouse fibroblast cells [63]. This result suggests that the LSm2-8 complex may act as a nuclear localization signal, but the cytoplasmic localization of the U6 snRNP is highly questionable. The actual function of the LSm 2-8 complex associated with U6 snRNA appears to be connected to U6 snRNP assembly and function. Mutants with decreased levels of LSm2-8 show splicing defects correlating with a reduced level of U6 snRNA. How the LSm2-8 complex affects U6 snRNP remains unclear. One possibility is that LSm proteins facilitate conformational rearrangements during the splicing cycle, U4/U6 annealing and formation of U4/U6/U5 tri-snRNP [64].
2.4. Role of LSm proteins in protecting mRNA 3` end termini from degradation
LSm proteins have additional roles apart from splicing. Yeast strains which lack LSm1-7p fail to grow at higher temperatures, and accumulate mRNA shortened at the 3` end by 20-30 nucleotides. The simplest model proposes that LSm1-7 complex binds to the mRNA and sterically inhibits endo and exo-nucleases. Nuclear LSm2-8 binds to the U6 snRNA 3` end, suggesting, that LSm2-8 protects the 3` end of U6 snRNA from degradation [65].
2.5. Role of LSm oligomer proteins in U8 snoRNP organization
U8 snoRNP is required for processing of 5.8S and 28S rRNAs, which together with the 5S rRNA build up the large ribosomal subunit. In Xenopus extract, LSm2, 3, 4, 6, 7, and 8 are bound as hetero hexamer to U8 snoRNA on the conserved third stem-loop sequences [66].
2.6. LSm oligomers as part of U7 snRNP
Maturation of the non-polyadenylated histone mRNAs 3' ends occurs by endonucleolytic cleavage mediated by U7 snRNP [67]. U7 snRNA contains a non-canonical Sm site. Purified U7 snRNP lacks D1 and D2 proteins but has LSm10 (14kDa) and LSm 11 (50kDa) instead [68].
2.7. LSm protein oligomers in mRNA degradation
Yeast two hybrid assays reveal multiple interactions between the eight LSm proteins, suggesting the existence of more than one LSm protein complex. Each human LSm protein is capable of interacting with multiple other LSm proteins and splicing factors, like prp24, prp4, and SmD1 [69]. Coprecipitation experiments demonstrated that LSm1p (LSmXp, is the nomenclature for yeast LSm proteins) together with LSm2p-LSm7p forms a new seven-subunit complex [70, 71]. The LSm complex LSm1-7 plays a role in mRNA degradation [72], and LSm2-8 has a role in the stabilization of U6 snRNP. These two protein complexes thus have very different functions. LSm1p mutants accumulate full length capped transcripts, but mutations on LSm1p do not stabilize mRNA containing premature stop codons, suggesting that the LSm1-7 complex is not involved in NMD [71]. The function of the LSm1-7 complex is most likely to interact with the mRNA substrate and accelerate decapping. Decapping is mediated by a decapping enzyme that is consisting of Dcp1a, Dcp1b, and the catalytic subunit Dcp2. The LSm1-7 proteins are localized in discrete cytoplasmic foci. The foci contain key decapping factors required for 5`-3` mRNA degradation. Coexpression of LSm proteins increases the number of foci. The cytoplasmic foci contain LSm1-7 proteins [73]. LSm1 and LSm8 are closely related to each other, and to the SmB protein. The 33 C terminal amino acids of LSm1 are necessary but not sufficient for proper cellular localization of hLSm1 [73]. Finally it has been demonstrated [73] that the foci are actual degradation centers, where mRNA degradation occurs. This suggests that the cytoplasm of cells is more organized than previously thought. Bacterial Hfq protein (pdb 1hk9) is able to chaperone RNA-RNA interactions similarly like LSm proteins ability to chaperone RNA/protein interactions and protect the 3' end of a transcript from exonucleolytic decay while encouraging degradation through other pathways [74].
2.8. LSm proteins in the processing of pre-tRNAs
It has been reported that depletion of LSm proteins in yeast leads to strong accumulation of unspliced tRNA species. The absence of LSm proteins most probably alters the pattern of processing intermediate [75].
2.9. LSm 2-7 complex associated with snR5
An LSm2-7 hexameric complex is found to be associated with snR5 in
A particularly interesting example of forming higher order complexes-oligomers is the Sm/LSm protein family (whose various complexes are described above), whose members are engaged in a variety of RNA processing events, forming complexes which differ sometimes only by one out of seven subunits. Another important aspect of the Sm/LSm protein family is that these proteins never occur in isolation; for proper functioning they require complex formation. Hence, the way to better understand Sm/LSm protein function is to study Sm/LSm complexes. It is difficult to determine the connection between the oligomeric state of a given protein and its function
Sequence comparisons of the yeast LSm protein family indicate that each canonical Sm protein has a corresponding LSm protein with the exception of SmB, which aligns almost equally well with LSm1 and LSm8. Based on sequence comparisons co expression vectors encoding the homologs of SmD1D2, LSm23, of SmD3B, LSm48, and of SmEFG, LSm567 were constructed and proteins were expressed in bacteria [77]. LSm4 and LSm1 were singly over expressed for the reconstitution of LSm1-7 [77].
Two heteroheptameric complexes LSm1-7 and LSm2-8 were reconstituted from two heterodimers and one heterotrimer in case of LSm2-8 (LSm2-3, LSm4-8, LSm5-6-7) and one heterotrimer, one heterodimer and two proteins singly expressed (LSm2-3, LSm5-6-7, LSm1, LSm8). Reconstitution of heteroheptamers was achieved by mixing of equimolar amounts of each appropriate protein at 37°C adding 4 M urea in order to disrupt higher order structures, because those proteins have tendency to oligomerize. After incubation, mixture of pure recombinant proteins was dialyzed against native buffer. Mixture was applied on to size exclusion chromatography, followed by the anion exchange chromatography. Last step in purification of homogenous heteroheptamers was size exclusion chromatography (peak profile shown on figure 11., and respective fractions were analyzed on polyacrylamide gel (shown of figure 12).
Negative stain electron micrographs show that reconstituted LSm2-8 has a ring-like architecture with a diameter of about 8 nm. The overall dimensions are similar to those previously observed for the native LSm2-8 complex isolated from HeLa cell nuclear extract (8 nm) and core snRNP domain [58]. The central cavity observed for the recombinant LSm2-8 complex is larger than in the native LSm2-8 complexes (3 vs. 2 nm, respectively). The LSm1-7 rings appear to be slightly smaller, measuring ~ 7 nm across with a pore diameter of less than 1.5 nm. Thus, recombinant LSm1-7 and LSm2-8 complexes are similar to one another and to the native Sm/LSm complexes at this level. In all LSm co-crystal structures solved with RNA oligonucleotides, the RNA molecules mainly wrap around the rim of the pore.
One of the methods which can be used for the identification and characterization of the RNA binding proteins is the electrophoretic mobility shift assay (EMSA). The basis of this method is the change in the electrophoretic mobility of a nucleic acid molecule upon binding to a protein or another molecule. Initially a labeled RNA, which contains the binding sequence, is incubated with a sample containing the RNA binding proteins and the mixture is then analyzed on a non-denaturing gel. The unbound RNA will have a characteristic electrophoretic mobility. Functionality of reconstituted LSm2-8 and LSm1-7 complexes has been demonstrated using this essay in vitro [77]. That oligomer complexes are functional in vivo has been shown [77], by injecting fluorescently labeled complexes into cytoplasm of living cells. They localized in expected cellular compartment, namely LSm 2-8 took nuclear localization and LSm1-7 complex remained in the cytoplasm. The structure-function relationships within the Sm/LSm protein family reflect three major interconnected features which illustrate why it is so important to solve the structures of Sm/LSm hetero-oligomeric complexes: First, Sm/LSm protein function is in general strictly dependent on complex formation. This holds for RNA binding, Sm/LSm-protein containing RNP biogenesis, interaction with non-Sm protein effector proteins, and RNA processing activity. The required interaction interfaces are apparently always three dimensional structural sites generated from several Sm/LSm subunits. High resolution structural information is clearly required to explain the molecular basis for this phenomenon. Second, exchange of only one or two subunits from one to another heterooligomeric (mostly heptameric) Sm/LSm complex changes its whole biology (see above). How such subtle structural changes can have these very large functional effects can only be addressed by solving the crystal structures of the respective complexes. Lastly, the ability of individual Sm/LSm proteins to assemble with different homologous binding partners to form architecturally very similar, yet functionally diverse complexes argues for a very fine balance between flexibility and specificity for the respective Sm-Sm interactions. Clearly, in order to understand the “molecular recognition code” governing the specificity balance mentioned above, more structural information on such interactions is indispensable. Recently crystal structure of Saccharomyces cerevisiae LSm2-8 complex bound to U6 snRNA had been determined (pdb code 4M7D) [80].
Acknowledgments
This work was supported by the grant No. 172001 from the Ministry of Science and Education, Republic of Serbia.
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