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![Independence of Amin complex formation on the presence of ATP and dissociation of Amin complex in the presence of ATP. (A) Time course of complex assembly for BS-PPT RNA (lanes 1 to 21) or for full-length PIP85.B pre-mRNA (lanes 22 to 27) in the presence of ATP (lanes 1 to 7 and 22 and 23), absence of ATP (lanes 8 to 14 and 24 and 25), or absence of ATP and presence of EDTA (lanes 15 to 21 and 26 and 27). RNAs were incubated for the times indicated as described in Materials and Methods, adjusted to 0.5 mg of heparin per ml, and separated on a native 4% polyacrylamide gel. Amin, minimal U2 snRNP complex; B/C, spliceosomal complexes B and C containing U2/4/5/6 snRNPs and pre-mRNA; A, prespliceosomal complex A containing U2 snRNP and pre-mRNA; H, nonspecific complexes; ء , a faster-migrating complex observed at low levels in the presence of ATP. (B [upper]) BS-PPT RNA was incubated in nuclear extract depleted of ATP for 20 min to form the Amin complex (lane 1) and then was chased with 1 nmol of cold competitor RNA per ml and reincubated for the time courses shown in either the absence (lanes 2 to 7) or presence (lanes 8 to 13) of ATP. Reaction mixtures then were adjusted to 0.5 mg of heparin per ml and loaded onto a native 4% polyacrylamide gel. (Lower) The RNA in samples from the reactions described above was analyzed on a 15% (19:1) 8 M urea gel. (C) BS-PPT RNA (lanes 1 to 8) and BS-PPT-3 Ј Exon RNA [RNA(146–234); lanes 9 to 16] were incubated in nuclear extract depleted of ATP for 20 min to form Amin or Amin-like complexes, respectively. Cold competitor RNA was added [cold BS-PPT RNA for lanes 1 to 8 or cold RNA(146–234) for lanes 9 to 16], and reaction mixtures were reincubated at 30 Њ C in the presence of ATP for the time courses indicated (lanes 3 to 8 and 11 to 16) or in the absence of ATP for 60 min (lanes 2 and 10). Lanes 1 and 9, no reincubation. Reactions were then analyzed as described above. (D) Kinetics of formation and dissociation of Amin complexes. For formation, complexes were assembled on BS-PPT RNA in the presence of ATP ( F [curve a ; as in panel A, lanes 1 to 7]) or in the absence of ATP ( s [curve b ; as in panel A, lanes 15 to 21]). For dissociation, Amin complexes were first assembled by incubation for 30 min in nuclear extract depleted of ATP and then were reincubated in the presence of ATP ( s [curve c ; as in panel B, lanes 8 to 13]). Relative complex formation was determined as the fraction of Amin complex relative to the input RNA. Polyacrylamide gels were quantitated with a Molecular Dynamics PhosphorImager and ImageQuant software, version 3.22.](profile/Patrick-Page-Mccaw/publication/14107526/figure/fig3/AS:282710124843008@1444414754225/Independence-of-Amin-complex-formation-on-the-presence-of-ATP-and-dissociation-of-Amin.png)
Independence of Amin complex formation on the presence of ATP and dissociation of Amin complex in the presence of ATP. (A) Time course of complex assembly for BS-PPT RNA (lanes 1 to 21) or for full-length PIP85.B pre-mRNA (lanes 22 to 27) in the presence of ATP (lanes 1 to 7 and 22 and 23), absence of ATP (lanes 8 to 14 and 24 and 25), or absence of ATP and presence of EDTA (lanes 15 to 21 and 26 and 27). RNAs were incubated for the times indicated as described in Materials and Methods, adjusted to 0.5 mg of heparin per ml, and separated on a native 4% polyacrylamide gel. Amin, minimal U2 snRNP complex; B/C, spliceosomal complexes B and C containing U2/4/5/6 snRNPs and pre-mRNA; A, prespliceosomal complex A containing U2 snRNP and pre-mRNA; H, nonspecific complexes; ء , a faster-migrating complex observed at low levels in the presence of ATP. (B [upper]) BS-PPT RNA was incubated in nuclear extract depleted of ATP for 20 min to form the Amin complex (lane 1) and then was chased with 1 nmol of cold competitor RNA per ml and reincubated for the time courses shown in either the absence (lanes 2 to 7) or presence (lanes 8 to 13) of ATP. Reaction mixtures then were adjusted to 0.5 mg of heparin per ml and loaded onto a native 4% polyacrylamide gel. (Lower) The RNA in samples from the reactions described above was analyzed on a 15% (19:1) 8 M urea gel. (C) BS-PPT RNA (lanes 1 to 8) and BS-PPT-3 Ј Exon RNA [RNA(146–234); lanes 9 to 16] were incubated in nuclear extract depleted of ATP for 20 min to form Amin or Amin-like complexes, respectively. Cold competitor RNA was added [cold BS-PPT RNA for lanes 1 to 8 or cold RNA(146–234) for lanes 9 to 16], and reaction mixtures were reincubated at 30 Њ C in the presence of ATP for the time courses indicated (lanes 3 to 8 and 11 to 16) or in the absence of ATP for 60 min (lanes 2 and 10). Lanes 1 and 9, no reincubation. Reactions were then analyzed as described above. (D) Kinetics of formation and dissociation of Amin complexes. For formation, complexes were assembled on BS-PPT RNA in the presence of ATP ( F [curve a ; as in panel A, lanes 1 to 7]) or in the absence of ATP ( s [curve b ; as in panel A, lanes 15 to 21]). For dissociation, Amin complexes were first assembled by incubation for 30 min in nuclear extract depleted of ATP and then were reincubated in the presence of ATP ( s [curve c ; as in panel B, lanes 8 to 13]). Relative complex formation was determined as the fraction of Amin complex relative to the input RNA. Polyacrylamide gels were quantitated with a Molecular Dynamics PhosphorImager and ImageQuant software, version 3.22.
Source publication
The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes.
We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex,
here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate...
Contexts in source publication
Context 1
... process was ATP dependent. To test whether this represented an active process, complexes were formed in the absence of ATP, challenged with excess cold competitor BS-PPT RNA, and reincubated either with or without the addition of ATP. During this reincubation, Amin complexes were dissociated in the presence of ATP, but not in the absence of ATP (Fig. 4B, upper panel; cf. lanes 8 to 13 with 2 to 7). This was not due to degradation of the RNA, which remained at similar levels throughout the incubations (Fig. 4B, lower panel; cf. lanes 8 to 13 with 2 to 7). The dissociation of complexes required both magnesium cation and hydrolyzable NTP. For example, AMP- PcP, AMP-cPP, or AMP-PnP could ...
Context 2
... BS-PPT RNA, and reincubated either with or without the addition of ATP. During this reincubation, Amin complexes were dissociated in the presence of ATP, but not in the absence of ATP (Fig. 4B, upper panel; cf. lanes 8 to 13 with 2 to 7). This was not due to degradation of the RNA, which remained at similar levels throughout the incubations (Fig. 4B, lower panel; cf. lanes 8 to 13 with 2 to 7). The dissociation of complexes required both magnesium cation and hydrolyzable NTP. For example, AMP- PcP, AMP-cPP, or AMP-PnP could not replace ATP, although other NTPs or deoxynucleoside triphosphates (dNTPs) could (data not shown). Thus, the Amin complex is a substrate for an NTP-dependent dissociation ...
Context 3
... both magnesium cation and hydrolyzable NTP. For example, AMP- PcP, AMP-cPP, or AMP-PnP could not replace ATP, although other NTPs or deoxynucleoside triphosphates (dNTPs) could (data not shown). Thus, the Amin complex is a substrate for an NTP-dependent dissociation activity that results in rapid dis- assembly of U2 snRNP-containing complexes (Fig. 4D, curve c). The level of complexes formed in the presence of ATP (curve a) is probably the sum of the two processes of complex formation without ATP (curve b) and of dissociation with ATP (curve c), indicating a dynamic assembly and disassembly of U2 snRNP ...
Context 4
... test whether the presence of additional sequences would alter the susceptibility of the complex to the dissociation ac- tivity, the stability of Amin complexes was compared to that of complexes formed on RNA additionally containing a 3 splice site and 3 exon [RNA(146-234); Fig. 4C]. Although this RNA ostensibly is similar to 3 half RNAs used to form A3 complex, it does not contain sequences 5 to the branch region and forms complexes in the absence of ATP, making this compar- ison possible. As before, preformed Amin complexes dissoci- ated rapidly when challenged with ATP and competitor BS- PPT RNA (lanes 2 to 8) ...
Citations
... Reduction of DHX15 stabilizes the ATP-independent interaction between U2 snRNP and a minimal intron Query et al. (1997) showed that a minimal RNA (A min substrate) containing only a branch point sequence followed by a PYT interacts with U2 snRNP in the absence of ATP to form the A min -complex. Notably, in the presence of ATP, the A min -complex is destabilized by an unknown entity (Newnham and Query 2001). ...
... In the absence of ATP, the expected A min -complex band forms in both extracts (Fig. 3A). Addition of ATP to the mock-depleted extract results in the loss of most A min -complex and the appearance of a faster migrating complex of unknown composition ( * ) that decreases over time (Query et al. 1997). In DHX15-depleted extracts with ATP, the A min -complex persists, indicating that DHX15 and/or a co-depleted factor is responsible for the ATP-dependent loss. ...
A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation and several RNA-dependent ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNPs engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 may have an expanded role in maintaining splicing fidelity.
... | Small RNAs: Base pairing to survive and function Small RNAs (sRNAs) are 50-500 nt long and are known to regulate gene expression in prokaryotes. The sRNAs are involved in regulating various cellular processes and provide necessary adaptation to growth/stress conditions (Livny & Waldor, 2007;Tomizawa et al., 1981;Wassarman, 2002 (Nilsen, 1994;Query et al., 1994Query et al., , 1996Query et al., , 1997Zhuang & Weiner, 1986) (Lima et al., 2003;Nowotny et al., 2005Nowotny et al., , 2007Wu et al., 1999) base-pairing interactions with their target mRNAs either in trans or in cis (Wassarman, 2002). The sRNAs ultimately affect translation by either perfect or imperfect and noncontiguous base-pairing interactions. ...
... Next U2 snRNA interacts with the mRNA through a sequence 5 0 -GUAGUA 3 0 that is complementary to the UACUAAC (UNYURAC) sequence present in the intron branch site (Query et al., 1994;Figure 4c). This interaction results in a branched RNA duplex where the adenosine is flipped out of the duplex (Query et al., 1996(Query et al., , 1997. ...
The human genome is pervasively transcribed and yet only a small fraction of these RNAs (less than 2%) are known to code for proteins. The vast majority of the RNAs are classified as noncoding RNAs (ncRNAs) and are further subgrouped as small (shorter than 200 bases) and long noncoding RNAs. The ncRNAs have been identified in all three domains of life and regulate diverse cellular processes through transcriptional and posttranscriptional gene regulation. Most of these RNAs work in conjunction with proteins forming a wide array of base pairing interactions. The determinants of these base pairing interactions are now becoming more evident and show striking similarities among the diverse group of ncRNAs. Here we present a mechanistic overview of pairing between RNA–RNA or RNA–DNA that dictates the function of ncRNAs; we provide examples to illustrate that ncRNAs work through shared evolutionary mechanisms that encompasses a guide–target interaction, involving not only classical Watson–Crick but also noncanonical Wobble and Hoogsteen base pairing. We also highlight the similarities in target selection, proofreading, and the ruler mechanism of ncRNA–protein complexes that confers target specificity and target site selection.
This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA‐Based Catalysis > RNA‐Mediated Cleavage
RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution
... Query et al. showed a minimal RNA (A min substrate) containing only a branch point sequence followed by a PYT interacts with U2 snRNP in the absence of ATP to form the A mincomplex (Query et al. 1997). Notably, in the presence of ATP, the A min -complex is destabilized by an unknown entity (Newnham and Query 2001). ...
... In the absence of ATP, the expected A min -complex band forms in both extracts (Fig. 3A). Addition of ATP to the mock-depleted extract results in the loss of most A min -complex and the appearance of a faster migrating complex of unknown composition (*) that decreases over time (Query et al. 1997). In DHX15-depleted extracts with ATP, the A min -complex persists, indicating that DHX15 and/or a co-depleted factor is responsible for the ATP-dependent loss. ...
A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation and several RNA-dependent-ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNP's engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 can have an expanded role in splicing.
... This recognition involves basepairing interactions between the 10 highly conserved nucleotides at the 5' end of U1 snRNA and the intron sequences of the pre-mRNA at the 5' splice site (G/GUAUGU in yeast or G/ GURAGU in vertebrates, where "/" represents the exon-intron junction and R stands for purine; Zhuang and Weiner, 1986). The U2 snRNP then recognizes the pre-mRNA branch site to form a pre-splicing complex called as complex A (Query et al., 1997). This recognition again involves a base-pairing interaction between a highly conserved sequence in U2 snRNA and the pre-mRNA branch site sequence (UACUAAC in yeast or YNYURAC in vertebrates, where Y, R, N, and the underlined adenosine represent pyrimidine, purine, any nucleotide, and the branch point nucleotide, respectively; Parker et al., 1987;Zhuang et al., 1989). ...
Small nuclear RNAs (snRNAs) are critical components of the spliceosome that catalyze the splicing of pre-mRNA. snRNAs are each complexed with many proteins to form RNA-protein complexes, termed as small nuclear ribonucleoproteins (snRNPs), in the cell nucleus. snRNPs participate in pre-mRNA splicing by recognizing the critical sequence elements present in the introns, thereby forming active spliceosomes. The recognition is achieved primarily by base-pairing interactions (or nucleotide-nucleotide contact) between snRNAs and pre-mRNA. Notably, snRNAs are extensively modified with different RNA modifications, which confer unique properties to the RNAs. Here, we review the current knowledge of the mechanisms and functions of snRNA modifications and their biological relevance in the splicing process.
... Biomolecules 2020, 10, 680 2 of 17 along with other related proteins [10]. SF3b complex is the major component of U2 small nuclear ribonucleoprotein (U2snRNP) and it is responsible for reinforcing the interaction between U2 snRNA and the branch site on pre-mRNA in the first transesterification reaction of splicing process [11][12][13][14]. ...
... The number of contacts between pre-determined groups is computed by using "gmx mindist" module implemented in GROMACS. We used 0.3 nm as the cut-off value to calculate the number of contacts between the mutated residue and pre-mRNA while 3 nm was used to calculate the number of contacts between side chain residues of p14 (residues 20-100) and pre-mRNA as p14 is defined in previous studies as a marker of branch point [12,13]. ...
Cancer is the second leading cause of death worldwide. The etiology of the disease has remained elusive, but mutations causing aberrant RNA splicing have been considered one of the significant factors in various cancer types. The association of aberrant RNA splicing with drug/therapy resistance further increases the importance of these mutations. In this work, the impact of the splicing factor 3B subunit 1 (SF3B1) K700E mutation, a highly prevalent mutation in various cancer types, is investigated through molecular dynamics simulations. Based on our results, K700E mutation increases flexibility of the mutant SF3B1. Consequently, this mutation leads to i) disruption of interaction of pre-mRNA with SF3B1 and p14, thus preventing proper alignment of mRNA and causing usage of abnormal 3’ splice site, and ii) disruption of communication in critical regions participating in interactions with other proteins in pre-mRNA splicing machinery. We anticipate that this study enhances our understanding of the mechanism of functional abnormalities associated with splicing machinery, thereby, increasing possibility for designing effective therapies to combat cancer at an earlier stage.
... The SF3b complex directly recognizes the BPS and surrounding intron sequences [29][30][31]. In the human B act complex, all seven components of the SF3b are structurally resolved, including SF3b155 (Hsh155 in S. cerevisi- [32]. ...
During each cycle of pre-mRNA splicing, the pre-catalytic spliceosome (B complex) is converted into the activated spliceosome (Bact complex), which has a well-formed active site but cannot proceed to the branching reaction. Here, we present the cryo-EM structure of the human Bact complex in three distinct conformational states. The EM map allows atomic modeling of nearly all protein components of the U2 small nuclear ribonucleoprotein (snRNP), including three of the SF3a complex and seven of the SF3b complex. The structure of the human Bact complex contains 52 proteins, U2, U5, and U6 small nuclear RNA (snRNA), and a pre-mRNA. Three distinct conformations have been captured, representing the early, mature, and late states of the human Bact complex. These complexes differ in the orientation of the Switch loop of Prp8, the splicing factors RNF113A and NY-CO-10, and most components of the NineTeen complex (NTC) and the NTC-related complex. Analysis of these three complexes and comparison with the B and C complexes reveal an ordered flux of components in the B-to-Bact and the Bact-to-B* transitions, which ultimately prime the active site for the branching reaction.
... SF3B4 is a subunit of the splicing factor 3b protein complex, which is a multi-protein complex that forms the U2 snRNP together with other units and is essential for the splicing process [240]. It is also known that the SF3b subunit binds to the pre-mRNA near the BP to reinforce U2 snRNP [241,242,243,244] and plays a key role in BP recognition during constitutive and alternative splicing [245,246,247]. There is also enrichment of the SF3A3 protein (red in Figure 5.5a) in the HepG2 cell line, which is another subunit of splicing factor 3 that interacts with the same U2 snRNP. ...
RNA-binding proteins (RBPs) are the primary regulators of all aspects of posttranscriptional gene regulation. In order to understand how RBPs perform their function, it is important to identify their binding sites. Recently, new techniques have been developed to employ high-throughput sequencing to study protein-RNA interactions in vivo, including the individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP). iCLIP identifies sites of protein-RNA crosslinking with nucleotide resolution in a transcriptome-wide manner. It is composed of over60steps,whichcanbemodified,butitisnotclearhowvariationsinthemethod affect the assignment of RNA binding sites. This is even more pertinent given that several variants of iCLIP have been developed. A central question of my research is how to correctly assign binding sites to RBPs using the data produced by iCLIP and similar techniques. I first focused on the technical analyses and solutions for the iCLIP method. I examinedcDNAdeletionsandcrosslink-associatedmotifstoshowthatthestartsof cDNAs are appropriate to assign the crosslink sites in all variants of CLIP, including iCLIP, eCLIP and irCLIP. I also showed that the non-coinciding cDNA-starts are caused by technical conditions in the iCLIP protocol that may lead to sequence constraintsatcDNA-endsinthefinalcDNAlibrary. Ialsodemonstratedtheimportance of fully optimizing the RNase and purification conditions in iCLIP to avoid thesecDNA-endconstraints. Next,IdevelopedCLIPo,acomputationalframework that assesses various features of iCLIP data to provide quality control standards which reveals how technical variations between experiments affect the specificity of assigned binding sites. I used CLIPo to compare multiple PTBP1 experiments produced by iCLIP, eCLIP and irCLIP, to reveal major effects of sequence constraintsatcDNA-endsorstarts,cDNAlengthdistributionandnon-specificcontaminants. Moreover, I assessed how the variations between these methods influence themechanisticconclusions. Thus,CLIPopresentsthequalitycontrolstandardsfor transcriptome-wide assignment of protein-RNA binding sites. I continued with analyses of RBP complexes by using data from spliceosomeiCLIP. This method simultaneously detects crosslink sites of small nuclear ribonucleoproteins (snRNPs) and auxiliary splicing factors on pre-mRNAs. I demonstratedthatthehighresolutionofspliceosome-iCLIPallowsfordistinctionbetween multiple proximal RNA binding sites, which can be valuable for transcriptomewide studies of large ribonucleoprotein complexes. Moreover, I showed that spliceosome-iCLIP can experimentally identify over 50,000 human branch points. In summary, I detected technical biases from iCLIP data, and demonstrated how such biases can be avoided, so that cDNA-starts appropriately assign the RNA binding sites. CLIPo analysis proved a useful quality control tool that evaluates data specificity across different methods, and I applied it to iCLIP, irCLIP and ENCODE eCLIP datasets. I presented how spliceosome-iCLIP data can be used to study the splicing machinery on pre-mRNAs and how to use constrained cDNAs from spliceosome-iCLIP data to identify branch points on a genome-wide scale. Taken together, these studies provide new insights into the field of RNA biology and can be used for future studies of iCLIP and related methods.
... The assembly of the spliceosome is started by the formation of complex A by the binding of U1 small nuclear ribonucleoprotein at the 5 0 SS and the binding of U2 small nuclear ribonucleoprotein to both the branch site and the 3 0 SS. Here the polypyrimidine tract is necessary for a correct identification and assembly of complex A. 39,40 Through the binding of U4, U5, and U6, complex B is formed, after which U1 and U4 are excluded again. 41 Finally, the association of cell division cycle 5 like (CDC5L) results in the active spliceosome. ...
... Human SF3b can be isolated as a stable complex that contains seven subunits: SF3b155, SF3b130, SF3b145, SF3b49, SF3b14b, p14/SF3b14a, and SF3b10 (Will et al., 2002). In the spliceosome, SF3b proteins contact the pre-mRNA at or near the BS, reinforcing the U2 snRNA/BS base-pairing interaction (Gozani et al., 1996Query et al., 1997;Will et al., 2001), and thereby play a key role in BS recognition and selection during constitutive and alternative splicing (Alsafadi et al., 2016;Corrionero et al., 2011;Darman et al., 2015). ...
... SF3b14a/p14 is regarded as a marker of the branchpoint adenosine, to which it crosslinks within prespliceosomes and subsequent spliceosomal complexes (Query et al., 1997;Will et al., 2001). BS3 crosslinking data for p14 ( Figure S3A; Table S2) allowed us to model its average position in the SF3b core. ...
SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155's HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155's HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform. SF3b155 residues, the mutation of which leads to cancer, contribute to the tertiary structure of the HEAT superhelix and its surface properties in the proximity of p14 and U2AF65. The molecular architecture of SF3b reveals the spatial organization of cancer-related SF3b155 mutations and advances our understanding of their effects on SF3b structure and function.
... The assembly of the spliceosome is started by the formation of complex A by the binding of U1 small nuclear ribonucleoprotein at the 5 0 SS and the binding of U2 small nuclear ribonucleoprotein to both the branch site and the 3 0 SS. Here the polypyrimidine tract is necessary for a correct identification and assembly of complex A. 39,40 Through the binding of U4, U5, and U6, complex B is formed, after which U1 and U4 are excluded again. 41 Finally, the association of cell division cycle 5 like (CDC5L) results in the active spliceosome. ...
The abnormal stimulation of the multiple signal transduction pathways downstream of the receptor tyrosine kinase mesenchymal-epithelial transition factor (cMET) promotes cellular transformation, tumor motility and invasion. Therefore, cMET has been the focus of prognostic and therapeutic studies in different tumor types, including non-small cell lung cancer (NSCLC). In particular, several cMET-inhibitors have been developed as innovative therapeutic candidates, and are currently under investigation in clinical trials. However, one of the challenges in establishing effective targeted treatments against cMET, remains the accurate identification of biomarkers for the selection of responsive subsets of patients.
