Assembly Of Ribonucleoprotein Complexes Research Articles

Isolation of Nucleoli

Nucleoli are now recognized as multi-functional nuclear domains involved in several fundamental cell processes such as ribosome biogenesis, regulation of the assembly of non-ribosomal ribonucleoprotein complexes, tRNA maturation, sequestration of protein, viral infection, and cellular ageing. Extensive proteomic analyses of these nucleolar domains after their purification have contributed to the description of their multiple biological functions. Because nucleoli are the largest and densest nuclear structures, they are easily amenable to purification from nuclei of cultured animal cells using the protocol described in this unit.

Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick?

Many neurogenetic disorders are caused by the mutation of ubiquitously expressed genes. One such disorder, spinal muscular atrophy, is caused by loss or mutation of the survival motor neuron1 gene (SMN1), leading to reduced SMN protein levels and a selective dysfunction of motor neurons. SMN, together with partner proteins, functions in the assembly of small nuclear ribonucleoproteins (snRNPs), which are important for pre-mRNA splicing. It has also been suggested that SMN might function in the assembly of other ribonucleoprotein complexes. Two hypotheses have been proposed to explain the molecular dysfunction that gives rise to spinal muscular atrophy (SMA) and its specificity to a particular group of neurons. The first hypothesis states that the loss of SMN's well-known function in snRNP assembly causes an alteration in the splicing of a specific gene (or genes). The second hypothesis proposes that SMN is crucial for the transport of mRNA in neurons and that disruption of this function results in SMA.

The long journey of actin and actin-associated proteins from genes to polysomes.

During gene expression, multiple regulatory steps make sure that alterations of chromatin structure are synchronized with RNA synthesis, co-transcriptional assembly of ribonucleoprotein complexes, transport to the cytoplasm and localized translation. These events are controlled by large multiprotein complexes commonly referred to as molecular machines, which are specialized and at the same time display a highly dynamic protein composition. The crosstalk between these molecular machines is essential for efficient RNA biogenesis. Actin has been recently proposed to be an important factor throughout the entire RNA biogenesis pathway as a component of chromatin remodeling complexes, associated with all eukaryotic RNA polymerases as well as precursor and mature ribonucleoprotein complexes. The aim of this review is to present evidence on the involvement of actin and actin-associated proteins in RNA biogenesis and propose integrative models supporting the view that actin facilitates coordination of the different steps in gene expression.

Differential 3′ splice site recognition of SMN1 and SMN2 transcripts by U2AF and U2 snRNP

Spinal Muscular atrophy is a prevalent genetic disease caused by mutation of the SMN1 gene, which encodes the SMN protein involved in assembly of small nuclear ribonucleoprotein (snRNP) complexes. A paralog of the gene, SMN2, cannot provide adequate levels of functional SMN because exon 7 is skipped in a significant fraction of the mature transcripts. A C to T transition located at position 6 of exon 7 is critical for the difference in exon skipping between SMN1 and SMN2. Here we report that this nucleotide difference results in increased ultraviolet light-mediated crosslinking of the splicing factor U2AF(65) with the 3' splice site of SMN1 intron 6 in HeLa nuclear extract. U2 snRNP association, analyzed by native gel electrophoresis, is also more efficient on SMN1 than on SMN2, particularly under conditions of competition, suggesting more effective use of limiting factors. Two trans-acting factors implicated in SMN regulation, SF2/ASF and hnRNP A1, promote and repress, respectively, U2 snRNP recruitment to both RNAs. Interestingly, depending on the transcript and the regulatory factor, the effects on U2 binding not always correlate with changes in U2AF(65) crosslinking. Furthermore, blocking recognition of a Tra2-beta1-dependent splicing enhancer located in exon 7 inhibits U2 snRNP recruitment without affecting U2AF(65) crosslinking. Collectively, the results suggest that both U2AF binding and other steps of U2 snRNP recruitment can be control points in SMN splicing regulation.

Metastasis Research Society–American Association for Cancer Research Joint Conference on Metastasis

By analogy, the study of metastasis is like a group of blind people studying an elephant. Each describes the pachyderm based on the part (s)he touches, but none comprehends the whole elephant because his/her exposure is limited. Likewise, the complexity of metastasis can only be appreciated when one

Architecture of the Pontin/Reptin Complex, Essential in the Assembly of Several Macromolecular Complexes

Pontin and reptin belong to the AAA+ family, and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 A. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared with the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different than previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins.

Inhibition of U snRNP assembly by a virus-encoded proteinase

It has been proposed that defects in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes could account for the death of motor neurons in spinal muscular atrophy (SMA). We discovered that infection of cultured cells with poliovirus results in the specific cleavage of the host factor Gemin3 by a virus-encoded proteinase, 2A(pro). Gemin3 is a component of the macromolecular SMN complex that mediates assembly of U snRNP complexes by aiding the heptameric oligomerization of Sm proteins onto U snRNAs. Using in vitro Sm core assembly assays, we found that lowering the intracellular amounts of Gemin3 by either poliovirus infection or small interfering RNA (siRNA)-mediated knockdown of Gemin3 resulted in reduced assembly of U snRNPs. Immunofluorescence analyses revealed a specific redistribution of Sm proteins from the nucleoplasm to the cytoplasmic periphery of the nucleus in poliovirus-infected cells. We propose that defects in U snRNP assembly may be shared features of SMA and poliomyelitis.

Quantitative Analysis of RNA‐mediated Protein–Protein Interactions in Living Cells by FRET

Specific assembly of ribonucleoprotein complexes is essential in controlling various cellular functions including gene regulation. Diverse scaffolds containing proteins or nucleic acids could play key roles in stabilizing specific ribonucleoprotein complexes by enhancing protein-protein or RNA-protein interactions. One such example is the assembly of active RNA polymerase II transcription elongation complex originating from HIV-1 long terminal repeat promoter that involves HIV-1-encoded Tat protein and viral mRNA structure, trans-activation responsive RNA, and human CyclinT1 which is a subunit of the positive transcription elongation factor complex b. By using genetically encoded fluorescent proteins fused with Tat and human CyclinT1, here we demonstrate that human CyclinT1 was diffused throughout the nucleus and specific interactions between Tat and human CyclinT1 altered the localization of human CyclinT1 to specific nuclear foci. We also found that trans-activation responsive RNA enhanced protein-protein interactions between human CyclinT1 and Tat in living cells. Our results highlights the importance of trans-activation responsive RNA as a scaffold for stable and high affinity assembly of two protein partners to form a regulatory switch essential in HIV-1 gene regulation. RNA-mediated assembly of ribonucleoprotein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication. Furthermore, our results suggest that Tat interactions with human CyclinT1 change the nuclear location of positive transcription elongation factor complex b to modulate positive transcription elongation factor complex b function and transcription of cellular genes.

The retrovirus RNA trafficking granule: from birth to maturity

Post-transcriptional events in the life of an RNA including RNA processing, transport, translation and metabolism are characterized by the regulated assembly of multiple ribonucleoprotein (RNP) complexes. At each of these steps, there is the engagement and disengagement of RNA-binding proteins until the RNA reaches its final destination. For retroviral genomic RNA, the final destination is the capsid. Numerous studies have provided crucial information about these processes and serve as the basis for studies on the intracellular fate of retroviral RNA. Retroviral RNAs are like cellular mRNAs but their processing is more tightly regulated by multiple cis-acting sequences and the activities of many trans-acting proteins. This review describes the viral and cellular partners that retroviral RNA encounters during its maturation that begins in the nucleus, focusing on important events including splicing, 3' end-processing, RNA trafficking from the nucleus to the cytoplasm and finally, mechanisms that lead to its compartmentalization into progeny virions.

Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen

Using the Capsuleen (Csul) methyltransferase as bait in the yeast two-hybrid system, we have identified a novel Drosophila protein containing multiple WD repeats and encoded by the valois (vsl) gene, which acts in pole plasm function. Vls is homologous to human MEP50, which forms a complex with the PRMT5 methyltransferase--the human homologue of Csul. We found that Vls localizes to the nuage in the nurse cells and to the pole plasm in the oocyte. Moreover vls is required for the synthesis and/or stability of Oskar and the localization of Tudor (Tud) in both the nuage and at the posterior pole of the oocyte. Furthermore, we show that Vls and a fragment of Tud interact directly in binding assay. As the PMRT5/MEP50 complex is involved in ribonucleoprotein complex assembly, we hypothesize that the Vls complex may play a similar function in assembling the nuage in nurse cells and the polar granules in the oocyte.

RNA Tertiary Structure and Cooperative Assembly of a Large Ribonucleoprotein Complex

The mechanisms that govern the ordered assembly of multiprotein ribonucleoprotein complexes are not well understood. The in vitro reconstitution of the small subunit of the bacterial ribosome provides a tractable system for the detailed study of ordered assembly. We present a quantitative thermodynamic description of the hierarchical binding of ribosomal proteins to 16S rRNA during assembly of the platform of the 30S ribosomal subunit. The binding of S8, S11, S15, and the S6:S18 heterodimer to the central domain of 16S rRNA has been measured both individually and in combination using isothermal titration calorimetry and gel mobility shift assays. Both enthalpy and free energy measurements demonstrate the cooperative binding of S15 and the S6:S18 heterodimer, but no cooperativity is observed for either S8 or S11. The results define a thermodynamic framework that describes cooperative platform assembly.

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain.

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop-loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop-loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.

Splicing of oskar RNA in the nucleus is coupled to its cytoplasmic localization.

oskar messenger RNA localization at the posterior pole of the Drosophila oocyte is essential for germline and abdomen formation in the future embryo. The nuclear shuttling proteins Y14/Tsunagi and Mago nashi are required for oskar mRNA localization, and they co-localize with oskar mRNA at the posterior pole of the oocyte. Their human homologues, Y14/RBM8 and Magoh, are core components of the exon-exon junction complex (EJC). The EJC is deposited on mRNAs in a splicing-dependent manner, 20-24 nucleotides upstream of exon-exon junctions, independently of the RNA sequence. This indicates a possible role of splicing in oskar mRNA localization, challenging the established notion that the oskar 3' untranslated region (3'UTR) is sufficient for this process. Here we show that splicing at the first exon-exon junction of oskar RNA is essential for oskar mRNA localization at the posterior pole. We revisit the issue of sufficiency of the oskar 3'UTR for posterior localization and show that the localization of unrelated transcripts bearing the oskar 3'UTR is mediated by endogenous oskar mRNA. Our results reveal an important new function for splicing: regulation of messenger ribonucleoprotein complex assembly and organization for mRNA cytoplasmic localization.

Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons.

Spinal muscular atrophy (SMA), a common autosomal recessive form of motoneuron disease in infants and young adults, is caused by mutations in the survival motoneuron 1 (SMN1) gene. The corresponding gene product is part of a multiprotein complex involved in the assembly of spliceosomal small nuclear ribonucleoprotein complexes. It is still not understood why reduced levels of the ubiquitously expressed SMN protein specifically cause motoneuron degeneration. Here, we show that motoneurons isolated from an SMA mouse model exhibit normal survival, but reduced axon growth. Overexpression of Smn or its binding partner, heterogeneous nuclear ribonucleoprotein (hnRNP) R, promotes neurite growth in differentiating PC12 cells. Reduced axon growth in Smn-deficient motoneurons correlates with reduced β-actin protein and mRNA staining in distal axons and growth cones. We also show that hnRNP R associates with the 3′ UTR of β-actin mRNA. Together, these data suggest that a complex of Smn with its binding partner hnRNP R interacts with β-actin mRNA and translocates to axons and growth cones of motoneurons.

High-resolution X-ray and NMR Structures of the SMN Tudor Domain: Conformational Variation in the Binding Site for Symmetrically Dimethylated Arginine Residues

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8 Å crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.

Studying the assembly of multicomponent protein and ribonucleoprotein complexes using surface plasmon resonance

The assembly of large macromolecular complexes is an important aspect of cellular organization and metabolism. Interactions involving such complexes in principle follow the same rules as the interactions between single proteins or other macromolecules and can therefore be investigated using similar approaches. We have developed protocols employing standard surface plasmon resonance technology that allow the investigation of interactions involving complex macromolecular structures. The principal experimental challenges arise from the possibility of parallel reactions where partially assembled or dissociated subcomplexes form a significant proportion of the molecule population and from an increased likelihood of unspecific binding events owing to the larger surface and statistically higher number of charged areas on multisubunit assemblies. Ways to experimentally avoid or, where this is not possible, to control for these complications are discussed.

Evidence for differential ribonucleoprotein complex assembly in vitro on the 5′-untranslated region of the human IGF-IR transcript

The type I insulin-like growth factor receptor (IGF-IR) plays a key role in the control of cellular proliferation and survival. The human IGF-IR transcript is characterized by an unusually long 1038 nucleotide 5′-untranslated region (5′-UTR). We hypothesized that the contribution of this complex 5′-untranslated RNA sequence to the post-transcriptional regulation of IGF-IR expression would involve a dynamic interplay between RNA structure and specific RNA-binding proteins. Here we have detected and characterized a diverse series of regulatory proteins binding the IGF-IR 5′-UTR under disparate conditions. One pair of proteins (∼42/38 kDa) binds readily to the intact 5′-UTR, which is predicted to adopt a highly base-paired, highly favorable (d G=−498 kcal/mol) three-domain structure. Another protein(s) (p20*) specifically induces formation of a novel RNA structure from within the initial 209 nucleotides of the nascent IGF-IR transcript, but fails to UV crosslink to this RNA sequence. A third group of proteins recognizes and binds the IGF-IR 5′-UTR under highly stringent conditions, but only after higher-ordered RNA structure has been disrupted. Our in vitro results indicate that the IGF-IR 5′-UTR may exist in at least three distinct states, and we propose that interconversion between these states might take place in vivo and differentially alter IGF-IR transcript utilization.

A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss of functional survival motor neuron 1 (SMN1) protein. This ubiquitously expressed protein is a component of a novel complex immunodetected in both the cytoplasm and the nucleus, which is associated with complexes involved in mRNA splicing, ribosome biogenesis and transcription. Here, we study a mutant protein corresponding to the N-terminal half of the protein that is encoded by the SMA frameshift mutation SMN 472del5. We show by confocal microscopy that the resulting mutant protein exhibits various distribution patterns in different transiently transfected COS cells. The mutant distributes into the nucleoplasm and/or the nucleolus, whereas the normal SMN protein accumulates at discrete nucleocytoplasmic dot-like structures previously named gems/Cajal bodies. The cell population with the nucleolar distribution is enriched upon treatment with mimosine, a synchronizing drug in late G(1) phase. Co-immunoprecipitation studies carried out on nuclear extracts reveal that both the endogenous SMN and mutant proteins are associated with complexes containing two major non-ribosomal nucleolar proteins, namely nucleolin and protein B23, and that the association is mediated, by among other things, RNA moieties. Both the association of the SMN protein with nucleolin-containing complexes and the nucleolin/B23 complex are disrupted in fibroblasts derived from a type I SMA patient harboring a homozygous SMN1 gene deletion. These findings suggest that altered assembly and/or stability of ribonucleoprotein complexes may contribute to the pathophysiological processes in SMA.

Kinetic studies of RNA–protein interactions using surface plasmon resonance

Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA–protein interactions. Protein–RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA–binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA–protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA–protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA–protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.

SMN tudor domain structure and its interaction with the Sm proteins.

Spinal muscular atrophy (SMA) is a common motor neuron disease that results from mutations in the Survival of Motor Neuron (SMN) gene. The SMN protein plays a crucial role in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes via binding to the spliceosomal Sm core proteins. SMN contains a central Tudor domain that facilitates the SMN-Sm protein interaction. A SMA-causing point mutation (E134K) within the SMN Tudor domain prevents Sm binding. Here, we have determined the three-dimensional structure of the Tudor domain of human SMN. The structure exhibits a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins. The E134K mutation does not disrupt the Tudor structure but affects the charge distribution within this binding site. An intriguing structural similarity between the Tudor domain and the Sm proteins suggests the presence of an additional binding interface that resembles that in hetero-oligomeric complexes of Sm proteins. Our data provide a structural basis for a molecular defect underlying SMA.