Large RNA-protein Complexes Research Articles

Unconventional Source of Neurotoxic Protein Aggregation from Organelle Off-Target Bax∆2 in Alzheimer's Disease.

Protein aggregates are a hallmark of Alzheimer's disease (AD). Extensive studies have focused on β-amyloid plaques and Tau tangles. Here, we illustrate a novel source of protein aggregates in AD neurons from organelle off-target proteins. Bax is a mitochondrial pore-forming pro-death protein. What happens to Bax if it fails to target mitochondria? We previously showed that a mitochondrial target-deficient alternatively spliced variant, Bax∆2, formed large cytosolic protein aggregates and triggered caspase 8-mediated cell death. Bax∆2 protein levels were low in most normal organs and the proteins were quickly degraded in cancer. Here, we found that 85% of AD patients had Bax∆2 required alternative splicing. Increased Bax∆2 proteins were mostly accumulated in neurons of AD-susceptible brain regions. Intracellularly, Bax∆2 aggregates distributed independently of Tau tangles. Interestingly, Bax∆2 aggregates triggered the formation of stress granules (SGs), a large protein-RNA complex involved in AD pathogenesis. Although the functional domains required for aggregation and cell death are the same as in cancer cells, Bax∆2 relied on SGs, not caspase 8, for neuronal cell death. These results imply that the aggregation of organelle off-target proteins, such as Bax∆2, broadens the scope of traditional AD pathogenic proteins that contribute to the neuronal stress responses and AD pathogenesis.

The SMN Complex at the Crossroad between RNA Metabolism and Neurodegeneration.

In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype.

Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Ribosomes are abundant, large RNA-protein complexes that are the sites of all protein synthesis in cells. Defects in ribosomal proteins (RPs), including proteoforms arising from genetic variations, alternative splicing of RNA transcripts, post-translational modifications and alterations of protein expression level, have been linked to a diverse range of diseases, including cancer and aging. Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets. In the present work, using E.coli 70S RPs as an example, we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry (MS) system, and then applied it to the HeLa 80S ribosome. The results were complemented by a bottom-up approach. In total, 50 out of 55 RPs were identified using the top-down approach. Among these, more than 30 RPs were found to have their N-terminal methionine removed. Additional modifications such as methylation, acetylation, and hydroxylation were also observed, and the modification sites were identified by bottom-up MS. In a HeLa 80S ribosomal sample, we identified 98 ribosomal proteoforms, among which multiple truncated 80S ribosomal proteoforms were observed, the type of information which is often overlooked by bottom-up experiments. Although their relevance to diseases is not yet known, the integration of top-down and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

SFPQ and NONO Proteins and Long Non-Coding NEAT1 RNA: Cellular Functions and Role in the HIV-1 Life Cycle

About 20 years ago, large RNA-protein complexes called paraspeckles were discovered in cell nuclei. The main components of these complexes are SFPQ and NONO proteins and the long noncoding RNA NEAT1. Later, these proteins were found free in the nucleus and even in the cytoplasm. The functions of NEAT1 and paraspeckle proteins are quite diverse including retention of RNAs subjected to multiple editing of adenosine to inosine in the nucleus, response to DNA damage, transcription regulation, control of mRNA stability, regulation of splicing, and participation in the cell response to viral infection. Thus, there are numerous, albeit contradictory, data on the involvement of NEAT1, SFPQ, and NONO in the HIV-1 replicative cycle at its various stages. Here, we tried to briefly review the main cellular functions of NEAT1 RNA and SFPQ and NONO proteins. The goal of this review was also to summarize and, if possible, systematize the existing data on their role in the HIV-1 life cycle.

Structural mechanism of GTPase-powered ribosome-tRNA movement

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

Statistical potentials for RNA-protein interactions optimized by CMA-ES

Characterizing RNA-protein interactions remains an important endeavor, complicated by the difficulty in obtaining the relevant structures. Evaluating model structures via statistical potentials is in principle straight-forward and effective. However, given the relatively small size of the existing learning set of RNA-protein complexes optimization of such potentials continues to be problematic. Notably, interaction-based statistical potentials have problems in addressing large RNA-protein complexes. In this study, we adopted a novel strategy with covariance matrix adaptation (CMA-ES) to calculate statistical potentials, successfully identifying native docking poses.

The Deubiquitinase USP39 Promotes ESCC Tumorigenesis Through Pre-mRNA Splicing of the mTORC2 Component Rictor.

Spliceosomes are large RNA-protein molecular complexes which mediate splicing of pre-mRNA in eukaryotic cells. Their function is frequently altered in cancer, providing opportunities for novel therapeutic approaches. The ubiquitin specific protease 39 (USP39) is a highly conserved deubiquitylation family member that plays an essential role in pre-mRNA splicing where it serves to assemble the mature spliceosome complex. Previous studies have reported that USP39 acts in an oncogenic manner where it contributes to cancer progression and predicts poor prognosis in various human tumor types. Here we report that USP39 is differentially upregulated in human esophageal squamous cell carcinoma (ESCC) and its expression is significantly associated with clinicopathological characteristics including differentiation status and TNM stage. We found the USP39 upregulation was maintained in ESCC cell lines where it functioned to promote cancer cell growth in vitro and in xenografts. RNA-seq analyses identified that mTOR pathway activation was affected by shRNA-mediated silencing of USP39. Subsequent biochemical analyses demonstrated that USP39 regulates the activity of mTORC2 by selectively enhancing the splicing and maturation of Rictor mRNA, although not other key mTORC components. Together, our report proposes USP39 as a biomarker and oncogenic factor in ESCC, with a potential for targeting the USP39/mTOR2/Rictor axis as a therapeutic strategy. Furthermore, our study adds ESCC to the list of cancers where USP39 contributes to tumorigenesis and progression.

Evidence of specialized ribosomes in osteoarthritic chondrocytes

Purpose: Osteoarthritis (OA) is the most common joint disorder, characterized by progressive loss and destruction of articular cartilage. Articular chondrocytes are highly specialized cells that produce and maintain the cartilage extracellular matrix (ECM). This requires continuous mRNA translation. Human ribosomes are large protein-RNA complexes containing 79 core proteins and 4 ribosomal RNAs (18S, 5.8S, 28S, and 5 rRNA). Recent studies demonstrate that ribosomes are not static translational machines but, in fact, dynamic and heterogeneous.

CPSF30-L-mediated recognition of mRNA m6A modification controls alternative polyadenylation of nitrate signaling-related gene transcripts in Arabidopsis

N6-methyladenosine (m6A), a ubiquitous internal modification of eukaryotic mRNAs, plays a vital role in almost every aspect of mRNA metabolism. However, there is little evidence documenting the role of m6A in regulating alternative polyadenylation (APA) in plants. APA is controlled by a large protein-RNA complex with many components, including CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30). In Arabidopsis, CPSF30 has two isoforms and the longer isoform (CPSF30-L) contains a YT512-B Homology (YTH) domain, which is unique to plants. In this study, we showed that CPSF30-L YTH domain binds to m6A in vitro. In the cpsf30-2 mutant, the transcripts of many genes including several important nitrate signaling-related genes had shifts in polyadenylation sites that were correlated with m6A peaks, indicating that these gene transcripts carrying m6A tend to be regulated by APA. Wild-type CPSF30-L could rescue the defects in APA and nitrate metabolism in cpsf30-2, but m6A-binding-defective mutants of CPSF30-L could not. Taken together, our results demonstrated that m6A modification regulates APA in Arabidopsis and revealed that the m6A reader CPSF30-L affects nitrate signaling by controlling APA, shedding new light on the roles of the m6A modification during RNA 3′-end processing in nitrate metabolism.

The 47S Pre-rRNA processing cascade of articular chondrocytes is impaired in an in vitro model of osteoarthritis

Purpose: Osteoarthritis (OA) is the most common degenerative joint disorder, characterized by progressive loss and destruction of articular cartilage. Chondrocytes are highly specialized cells with primary function of producing and maintaining the extracellular matrix (ECM). This requires continuous translation of mRNAs by ribosomes. Mammalian ribosomes are large protein-RNA complexes containing 79 protein subunits and 4 ribosomal RNAs (18S, 5.8S, 28S, and 5 rRNAs). In the human system, three ribosomal RNAs, 18S, 5.8S, and 28S, arise from a single 47S pre-rRNA transcript. Maturation of these rRNAs takes place by a cascade of exo- and endo-ribonucleolytic cleavages of the primary 47S transcript. A limited number of small nucleolar RNAs (snoRNAs), such as RMRP are known to guide endoribonucleolytic cleavages of the 47S pre-rRNA transcript. Here, we hypothesized that 47S pre-rRNA processing is impaired in osteoarthritic articular chondrocytes, and contributes to OA progression. Methods: Primary human knee OA articular chondrocytes (pool of four donors, p2, seeding density 30,000 cells/cm2) were cultured in DMEM/F12, 10% FCS, 1% P/S, 1% NEAA and exposed to knee OA synovial fluid (OA-SF, 20% (v/v), pool of five donors) for 1-14 days. Expression levels of chondrocytes phenotypic marker genes (COL2A1, ACAN, COL10A1, COL1A1), selected 47S processing intermediates and RMRP snoRNA were measured by qPCR. Statistical significance was assessed by two-way ANOVA, Bonferroni post-tests, *p<0.05, **p<0.01, ***p<0.001 Results: To test our hypothesis, we set-up an OA-mimicking in vitro model using human primary articular chondrocytes exposed to osteoarthritic SF (OA-SF) for 1 to 14 days. The expression of chondrogenic marker genes COL2A1 (Figure 1A) and ACAN, as well as hypertrophic marker gene COL10A1, were significantly decreased immediately after the first day of treatment with OA-SF. The expression of COL1A1 (Figure. 1B), a marker of chondrocyte dedifferentiation, increased significantly upon OA-SF treatment in a time-dependent manner. To evaluate the 47S pre-rRNA processing cascade, primers covering selected cleavage sites (01, 2 and 4a) in the 47S transcript were designed (Figure 1C). Upon OA-SF treatment, we observed accelerated cleavage at site 01 within the 5’ ETS region, and site 2 within the ITS1. Cleavage at site 2 separates pre-18S and pre-5.8S-28S intermediates and it is guided by snoRNA RMRP, expression of which was increased upon OA-SF treatment. On the contrary, cleavage at site 4a within the ITS2 region, generating separated pre-5.8S and pre-28S precursors, was hindered in our model (Figure 1D). These results demonstrate that the dynamics of the 47S primary transcript processing is altered in our OA-mimicking in vitro model, showing accelerated processing towards mature 18S rRNA, and hindered processing towards mature 5.8S and 28S rRNAs. Conclusions: During OA, the chondrocyte’s environment changes dramatically, fueling a gradual change of the chondrocyte’s phenotype. In response to OA onset and progression, chondrocytes can adopt a variety of different OA-associated phenotypes ranging from proliferative, pre-hypertrophic, hypertrophic to a fibroblast-like. Upon OA-SF treatment we observed a progressive decrease in expression of COL2A1, ACAN, as well as COL10A1 genes, while expression of the dedifferentiation marker gene COL1A1 was significantly increased. Such an expression pattern is typical for dedifferentiating chondrocytes, and it indicates that chondrocytes treated with OA-SF are predominantly acquiring a fibroblast-like phenotype. Our results demonstrate alterations in the 47S pre-rRNA processing cascade, suggesting that 47S processing is vulnerable to changes in the chondrocyte’s extracellular environment. That can potentially lead to OA pathobiologically important alterations in ribosome biogenesis with consequences for the chondrocyte’s proteome. Future experiments are focussing on the validation of these findings by northern blotting and detailed unbiased mapping of the 47S processing cascade using RNA sequencing technology.

Structural basis for DEAH-helicase activation by G-patch proteins

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA-protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch-mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

SF3b4: A Versatile Player in Eukaryotic Cells.

Spliceosomes are large protein-RNA complexes regulating pre-mRNA processing in eukaryotes. SF3b4 encodes a core subunit of the U2-type spliceosome, loss- or gain-of-function of which often associates with abnormal cell growth, leading to tumorigenesis. Homologs of SF3b4 in other phyla are also essential. In this review, we summarize recent findings on the function of SF3b4. Importantly, we highlight the versatile roles of SF3b4, not only as a component for pre-mRNA splicing, but also as a regulator for transcription, translation, and cell signaling. Recent studies of SF3b4 homologs in different species across evolution will facilitate a better understanding of human diseases caused by the malfunction of SF3b4, such as Nager syndrome (NS) and cancer, in the future.

Retinal Pigment Epithelial Cells: The Unveiled Component in theEtiology of Prpf Splicing Factor-Associated Retinitis Pigmentosa.

Pre-mRNA splicing is a critical step in RNA processing in all eukaryotic cells. It consists of introns removal and requires the assembly of a large RNA-protein complex called the spliceosome. This complex of small nuclear ribonucleoproteins is associated with accessory proteins from the pre-mRNA processing factor (PRPF) family. Mutations in different splicing factor-encoding genes were identified in retinitis pigmentosa (RP) patients. A surprising feature of these ubiquitous factors is that the outcome of their alteration is restricted to the retina. Because of their high metabolic demand, most studies focused on photoreceptors dysfunction and associated degeneration. However, cells from the retinal pigment epithelium (RPE) are also crucial to maintaining retinal homeostasis and photoreceptor function. Moreover, mutations in RPE-specific genes are associated with some RP cases. Indeed, we identified major RPE defects in Prpf31-mutant mice: circadian rhythms of both photoreceptor outer segments (POS) phagocytosis and retinal adhesion were attenuated or lost, leading to ultrastructural anomalies and vacuoles. Taken together, our published and ongoing data suggest that (1) similar molecular events take place in human and mouse cells and (2) these functional defects generate various stress processes.

Solid-State NMR Spectroscopy of RNA.

RNA structure is essential to understand RNA function and regulation in cellular processes. RNA acts either in isolation or as part of a complex with proteins. Both isolated and protein-complexed RNA represent a challenge for structural biology, due to the complexity of its conformational space and intrinsic dynamics. NMR, with its capability to cope with dynamic structures, is the optimal technique to study RNA conformation, ideally in solution. However, RNA-protein complexes are often very large, which limits the application of solution-state NMR. In this chapter we describe the methodology that we developed to determine the structure of RNA by solid-state NMR. Solid-state NMR is not limited by large molecular weights and can be optimally applied to study both the RNA and the protein components of large RNA-protein complexes. We review the methods for resonance assignments, collection of RNA-specific distance restraints, as well as detection of protein-RNA interfaces.

Role of N-myristoylation in stability and subcellular localization of the CLPABP protein

Cardiolipin and phosphatidic acid-binding protein (CLPABP) controls the stability of the mRNA harboring an AU-rich element (ARE) in the 3′ UTR with the help of the RNA stabilizer, human antigen R (HuR). Although CLPABP is localized on the mitochondrial surface as a large protein-RNA complex, its precise role is not yet known. Recently, CLPABP was identified as an N-myristoylated protein. Here, we demonstrate the effects of N-myristoylation on the functions of CLPABP. In the present study, compared to the wild-type protein that possessed the “MG” motif at the N-terminus for N-myristoylation, the mutant CLPABP protein that lacked N-myristoylation modification site was unstable. Furthermore, the expression of the G/A mutant of CLPABP, which lacked N-myristoylation site, induced morphological alterations in mitochondria. Because pleckstrin homology domain-deleted mutant, which was fused with the N-myristoylation site derived from intact CLPABP, could not colocalize with mitochondria, N-myristoylation of CLPABP was predicted to affect its stability onto the mitochondrial membrane rather than its subcellular localization.

Heterozygous HNRNPU variants cause early onset epilepsy and severe intellectual disability.

Pathogenic variants in genes encoding subunits of the spliceosome are the cause of several human diseases, such as neurodegenerative diseases. The RNA splicing process is facilitated by the spliceosome, a large RNA-protein complex consisting of small nuclear ribonucleoproteins (snRNPs), and many other proteins, such as heterogeneous nuclear ribonucleoproteins (hnRNPs). The HNRNPU gene (OMIM *602869) encodes the heterogeneous nuclear ribonucleoprotein U, which plays a crucial role in mammalian development. HNRNPU is expressed in the fetal brain and adult heart, kidney, liver, brain, and cerebellum. Microdeletions in the 1q44 region encompassing HNRNPU have been described in patients with intellectual disability (ID) and other clinical features, such as seizures, corpus callosum abnormalities (CCA), and microcephaly. Recently, pathogenic HNRNPU variants were identified in large ID and epileptic encephalopathy cohorts. In this study, we provide detailed clinical information of five novels and review two of the previously published individuals with (likely) pathogenic de novo variants in the HNRNPU gene including three non-sense and two missense variants, one small intragenic deletion, and one duplication. The phenotype in individuals with variants in HNRNPU is characterized by early onset seizures (6/7), severe ID (6/6), severe speech impairment (6/6), hypotonia (6/7), and central nervous system (CNS) (5/6), cardiac (4/6), and renal abnormalities (3/4). In this study, we broaden the clinical and mutational HNRNPU-associated spectrum, and demonstrate that heterozygous HNRNPU variants cause epilepsy, severe ID with striking speech impairment and variable CNS, cardiac, and renal anomalies.

The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript.

We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA "footprint" located on the 5' half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.

Antagonizing effect of CLPABP on the function of HuR as a regulator of ARE-containing leptin mRNA stability and the effect of its depletion on obesity in old male mouse

Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein–RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3′UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.

The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

Ribosomes are abundant, large RNA–protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease.

The organization and contribution of helicases to RNA splicing.

Splicing is an essential step of gene expression. It occurs in two consecutive chemical reactions catalyzed by a large protein-RNA complex named the spliceosome. Assembled on the pre-mRNA substrate from five small nuclear proteins, the spliceosome acts as a protein-controlled ribozyme to catalyze the two reactions and finally dissociates into its components, which are re-used for a new round of splicing. Upon following this cyclic pathway, the spliceosome undergoes numerous intermediate stages that differ in composition as well as in their internal RNA-RNA and RNA-protein contacts. The driving forces and control mechanisms of these remodeling processes are provided by specific molecular motors called RNA helicases. While eight spliceosomal helicases are present in all organisms, higher eukaryotes contain five additional ones potentially required to drive a more intricate splicing pathway and link it to an RNA metabolism of increasing complexity. Spliceosomal helicases exhibit a notable structural diversity in their accessory domains and overall architecture, in accordance with the diversity of their task-specific functions. This review summarizes structure-function knowledge about all spliceosomal helicases, including the latter five, which traditionally are treated separately from the conserved ones. The implications of the structural characteristics of helicases for their functions, as well as for their structural communication within the multi-subunits environment of the spliceosome, are pointed out.