Sequence and structural evolution of the KsgA/Dim1 methyltransferase family

<p>Abstract</p> <p>Background</p> <p>One of the 60 or so genes conserved in all domains of life is the <it>ksgA/dim1 </it>orthologous group. Enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. Despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions. For example, in <it>S. cerevisiae </it>Dim1 performs two distinct functions in ribosome biogenesis, while human mtTFB is not only an rRNA methyltransferase in the mitochondria but also a mitochondrial transcription factor. Thus, these proteins offer an unprecedented opportunity to study evolutionary aspects of structure/function relationships, especially with respect to our recently published work on the binding mode of a KsgA family member to its 30S subunit substrate. Here we compare and contrast KsgA orthologs from bacteria, eukaryotes, and mitochondria as well as the paralogous ErmC enzyme.</p> <p>Results</p> <p>By using structure and sequence comparisons in concert with a unified ribosome binding model, we have identified regions of the orthologs that are likely related to gains of function beyond the common methyltransferase function. There are core regions common to the entire enzyme class that are associated with ribosome binding, an event required in rRNA methylation activity, and regions that are conserved in subgroups that are presumably related to non-methyltransferase functions.</p> <p>Conclusion</p> <p>The ancient protein KsgA/Dim1 has adapted to cellular roles beyond that of merely an rRNA methyltransferase. These results provide a structural foundation for analysis of multiple aspects of ribosome biogenesis and mitochondrial transcription.</p

Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells

Analysis of the EIAV Rev-Responsive Element (RRE) Reveals a Conserved RNA Motif Required for High Affinity Rev Binding in Both HIV-1 and EIAV

A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies

Lower Cancer Incidence in Amsterdam-I Criteria Families Without Mismatch Repair Deficiency: Familial Colorectal Cancer Type X

Approximately 60% of families that meet the Amsterdam-I criteria (AC-I) for hereditary nonpolyposis colorectal cancer (HNPCC) have a hereditary abnormality in a DNA mismatch repair (MMR) gene. Cancer incidence in AC-I families with MMR gene mutations is reported to be very high, but cancer incidence for individuals in AC-I families with no evidence of an MMR defect is unknown

MC1R variants in childhood and adolescent melanoma: a retrospective pooled analysis of a multicentre cohort.

BACKGROUND: Germline variants in the melanocortin 1 receptor gene (MC1R) might increase the risk of childhood and adolescent melanoma, but a clear conclusion is challenging because of the low number of studies and cases. We assessed the association of MC1R variants with childhood and adolescent melanoma in a large study comparing the prevalence of MC1R variants in child or adolescent patients with melanoma to that in adult patients with melanoma and in healthy adult controls. METHODS: In this retrospective pooled analysis, we used the M-SKIP Project, the Italian Melanoma Intergroup, and other European groups (with participants from Australia, Canada, France, Greece, Italy, the Netherlands, Serbia, Spain, Sweden, Turkey, and the USA) to assemble an international multicentre cohort. We gathered phenotypic and genetic data from children or adolescents diagnosed with sporadic single-primary cutaneous melanoma at age 20 years or younger, adult patients with sporadic single-primary cutaneous melanoma diagnosed at age 35 years or older, and healthy adult individuals as controls. We calculated odds ratios (ORs) for childhood and adolescent melanoma associated with MC1R variants by multivariable logistic regression. Subgroup analysis was done for children aged 18 or younger and 14 years or younger. FINDINGS: We analysed data from 233 young patients, 932 adult patients, and 932 healthy adult controls. Children and adolescents had higher odds of carrying MC1R r variants than did adult patients (OR 1·54, 95% CI 1·02-2·33), including when analysis was restricted to patients aged 18 years or younger (1·80, 1·06-3·07). All investigated variants, except Arg160Trp, tended, to varying degrees, to have higher frequencies in young patients than in adult patients, with significantly higher frequencies found for Val60Leu (OR 1·60, 95% CI 1·05-2·44; p=0·04) and Asp294His (2·15, 1·05-4·40; p=0·04). Compared with those of healthy controls, young patients with melanoma had significantly higher frequencies of any MC1R variants. INTERPRETATION: Our pooled analysis of MC1R genetic data of young patients with melanoma showed that MC1R r variants were more prevalent in childhood and adolescent melanoma than in adult melanoma, especially in patients aged 18 years or younger. Our findings support the role of MC1R in childhood and adolescent melanoma susceptibility, with a potential clinical relevance for developing early melanoma detection and preventive strategies. FUNDING: SPD-Pilot/Project-Award-2015; AIRC-MFAG-11831

Assembly of the 30S ribosomal subunit: Positioning ribosomal protein S13 in the S7 assembly branch

Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 Å away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3′ major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data

Ribosomal small subunit assembly : a comparative approach

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2015.Ribonucleoproteins (RNPs) are present in all branches of life and perform vital functions required for cell viability. One such RNP is the bacterial ribosome. Composed of three rRNA and over 50 r-proteins this macromolecular complex must be assembled accurately and efficiently for proper translation and subsequently cell viability. Fascinatingly, the Small SubUnit (SSU) of E. coli can be reconstituted in vitro from the core components of 16S rRNA and 20 r-proteins. This has given vital information on the form and function of the SSU, but the majority is from E. coli. To expand the current knowledge of SSU assembly, we have studied in vitro SSU assembly of two thermophilic bacteria, G. kaustophilus and T. thermophilus in two different ways. We first looked to compare the temperature dependent assembly of SSUs from E. coli, G. kaustophilus and T. thermophilus. We found, in all three organisms, that at least two, distinct temperature dependent intermediates formed. These intermediate RNPs are incapable of proper translation indicating that there may be a conserved pathway within the 16S rRNA and TP30 components which help to ensure that improperly assembled SSUs remain unable to enter the translation cycle. To further our understanding of the rRNA and r-protein interactions within SSUs from E. coli and the thermophilic bacteria G. kaustophilus and T. thermophilus, we used a system of hybrid reconstitution. This study reconstituted 16S rRNA from one organism and r-proteins from another to form SSUs. We show that the temperature dependent assembly of hybrid SSUS is dependent on the optimum growth and reconstitution temperature of organism from which the r-proteins are purified. This indicates the proper folding and assembly of at least one r-protein drives the temperature dependence of assembly. These studies do a great deal to expand on the current knowledge on SSU assembly in a diverse range of bacteria as well our understanding of formation of RNPs in general

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25–26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 5′ terminus. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation

A systematic analysis of ribosomal small subunit biogenesis in wild-type E. coli

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2014.Ribonucleoproteins (RNPs) perform diverse biological functions, from catalysis to regulation of gene expression. Ribosomes are complex RNPs that synthesize proteins in all living organisms. Ribosome assembly in bacteria has been studied using in vitro reconstitution experiments for over five decades. In vitro reconstitution is not truly representative of ribosome assembly as in vivo ribosome biogenesis requires transcription, processing and modification of 5000 nucleotides of ribosomal RNA (rRNA), and is aided by several auxiliary factors, which are generally not included in in vitro reconstitution. In vivo ribosome biogenesis in bacteria is poorly understood because it is a complex, efficient and asynchronous process with only a small pool of intermediates present in wild-type cells at any given time. This thesis presents novel techniques and findings on the assembly of ribosomal small subunit (SSU) in wild-type E. coli under optimal growth conditions. To study SSU assembly in E. coli, we developed RNP affinity purification techniques to isolate and characterize in vivo formed SSU intermediates. These approaches took advantage of the regions in precursor 16S rRNA (pre-16S rRNA, leader and trailer) that are components of the pre- rRNA and SSU intermediates but are absent in mature SSUs or ribosomes. An RNA affinity tag was inserted in pre-16S rRNA at different positions between various nucleolytic cleavage sites, allowing systematic purification of different intermediates and mapping of the assembly cascade. The first precursor of 16S rRNA (17S rRNA) is the major platform for SSU biogenesis in vivo. Structural probing demonstrated that these purified 17S rRNA containing SSU intermediates had diverse architectures representing early to late stages of SSU biogenesis. These intermediates are likely incapable of translation as the regions of 16S rRNA involved in translation showed altered structure compared to the corresponding regions in mature SSUs suggesting there are checkpoints that prevent immature subunits to enter the translation cycle. The three pre-SSUs exhibited differential association of ribosomal proteins and known auxiliary factors and thus revealed multiple pathways for these processes during SSU biogenesis. Several novel and putative auxiliary factors were also identified using proteomic analysis, and preliminary characterization had demonstrated their role in SSU biogenesis. Additionally, substrates for two 16S rRNA modification enzymes were partially characterized. Our results indicate that there are multiple pathways for different biogenesis processes and SSU assembly occurs largely on 17S rRNA. Final 17S rRNA processing events happen late in the biogenesis cascade and follow multiple pathways with independent 5′ end or 3′ end maturation of 17S rRNA. These findings allow the first integration of rRNA processing events with conformational changes, rRNA modification, r-proteins association and auxiliary factor action during ribosomal SSU biogenesis in wild-type bacteria