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1.
Dev Cell ; 58(17): 1593-1609.e9, 2023 09 11.
Article in English | MEDLINE | ID: mdl-37473757

ABSTRACT

Translational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2'-O-methylation (2'-O-me) of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here, we examine changes in rRNA 2'-O-me during mouse brain development and tri-lineage differentiation of human embryonic stem cells (hESCs). We find distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. We identify a methylation site impacting neuronal differentiation. Modulation of its methylation levels affects ribosome association of the fragile X mental retardation protein (FMRP) and is accompanied by an altered translation of WNT pathway-related mRNAs. Together, these data identify ribosome heterogeneity through rRNA 2'-O-me during early development and differentiation and suggest a direct role for ribosomes in regulating translation during cell fate acquisition.


Subject(s)
RNA, Ribosomal , Ribosomes , Humans , Animals , Mice , Methylation , Ribosomes/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Cell Differentiation , Neurogenesis/genetics , Ribosomal Proteins/metabolism
2.
Biomolecules ; 12(2)2022 01 20.
Article in English | MEDLINE | ID: mdl-35204665

ABSTRACT

Twenty new compounds, targeting CYP17A1, were synthesized, based on our previous work on a benzimidazole scaffold, and their biological activity evaluated. Inhibition of CYP17A1 is an important modality in the treatment of prostate cancer, which remains the most abundant cancer type in men. The biological assessment included CYP17A1 hydroxylase and lyase inhibition, CYP3A4 and P450 oxidoreductase (POR) inhibition, as well as antiproliferative activity in PC3 prostate cancer cells. The most potent compounds were selected for further analyses including in silico modeling. This combined effort resulted in a compound (comp 2, IC50 1.2 µM, in CYP17A1) with a potency comparable to abiraterone and selectivity towards the other targets tested. In addition, the data provided an understanding of the structure-activity relationship of this novel non-steroidal compound class.


Subject(s)
Enzyme Inhibitors , Prostatic Neoplasms , Steroid 17-alpha-Hydroxylase , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Male , PC-3 Cells , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/enzymology , Steroid 17-alpha-Hydroxylase/antagonists & inhibitors , Structure-Activity Relationship
3.
Blood ; 139(2): 245-255, 2022 01 13.
Article in English | MEDLINE | ID: mdl-34359076

ABSTRACT

Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed, because current treatments do not cure most patients with AML. We report a domain-focused, kinome-wide CRISPR-Cas9 screening that identified protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. Loss of RIOK2 led to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, the ATPase function of RIOK2 was necessary for cell survival. When a small-molecule inhibitor was used, pharmacological inhibition of RIOK2 similarly led to loss of protein synthesis and apoptosis and affected leukemic cell growth in vivo. Our results provide proof of concept for targeting RIOK2 as a potential treatment of patients with AML.


Subject(s)
Leukemia, Myeloid, Acute , Protein Kinase Inhibitors , Animals , Mice , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , CRISPR-Cas Systems , Gene Expression Regulation, Leukemic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Molecular Targeted Therapy , Protein Biosynthesis/drug effects , Protein Kinase Inhibitors/pharmacology
4.
Nat Struct Mol Biol ; 28(11): 889-899, 2021 11.
Article in English | MEDLINE | ID: mdl-34759377

ABSTRACT

Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms and can arise from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2'-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signaling pathways, such as MYC oncogene expression. Ablation of one prominent methylation resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2'-O-methylation can give rise to ribosomes with specialized function. This suggests a broader mechanism where the specific regulation of rRNA modification patterns fine tunes translation.


Subject(s)
Protein Biosynthesis/physiology , Proto-Oncogene Proteins c-myc/genetics , RNA Processing, Post-Transcriptional/physiology , RNA, Ribosomal/metabolism , Ribosomes/metabolism , Cell Line, Tumor , HeLa Cells , Humans , Methylation , Protein Processing, Post-Translational/physiology , Proto-Oncogene Proteins c-myc/biosynthesis , RNA, Messenger/genetics
5.
Int J Mol Sci ; 21(14)2020 Jul 09.
Article in English | MEDLINE | ID: mdl-32660148

ABSTRACT

The current study presents the design, synthesis, and evaluation of novel cytochrome P450 17A1 (CYP17A1) ligands. CYP17A1 is a key enzyme in the steroidogenic pathway that produces androgens among other steroids, and it is implicated in prostate cancer. The obtained compounds are potent enzyme inhibitors (sub µM) with antiproliferative activity in prostate cancer cell lines. The binding mode of these compounds is also discussed.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Prostatic Neoplasms/drug therapy , Steroid 17-alpha-Hydroxylase/antagonists & inhibitors , Androgens/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Male , PC-3 Cells , Prostatic Neoplasms/metabolism
6.
J Extracell Vesicles ; 8(1): 1578116, 2019.
Article in English | MEDLINE | ID: mdl-30815237

ABSTRACT

The prevalent porcine helminth, Ascaris suum, compromises pig health and reduces farm productivity worldwide. The closely related human parasite, A. lumbricoides, infects more than 800 million people representing a disease burden of 1.31 million disability-adjusted life years. The infections are often chronic in nature, and the parasites have a profound ability to modulate their hosts' immune responses. This study provides the first in-depth characterisation of extracellular vesicles (EVs) from different developmental stages and body parts of A. suum and proposes the role of these vesicles in the host-parasite interplay. The release of EVs from the third- (L3) and fourth-stage (L4) larvae and adults was demonstrated by transmission electron microscopy (TEM), and sequencing of EV-derived RNA identified a number of microRNAs (miRNAs) and transcripts of potential host immune targets, such as IL-13, IL-25 and IL-33, were identified. Furthermore, proteomics of EVs identified several proteins with immunomodulatory properties and other proteins previously shown to be associated with parasite EVs. Taken together, these results suggest that A. suum EVs and their cargo may play a role in host-parasite interactions. This knowledge may pave the way to novel strategies for helminth infection control and knowledge of their immune modulatory potential.

7.
Biomolecules ; 8(4)2018 10 30.
Article in English | MEDLINE | ID: mdl-30380771

ABSTRACT

In eukaryotes, 18S, 5.8S, and 28S rRNAs are transcribed as precursor molecules that undergo extensive modification and nucleolytic processing to form the mature rRNA species. Central in the process are the small nucleolar RNAs (snoRNAs). The majority of snoRNAs guide site specific chemical modifications but a few are involved in defining pre-rRNA cleavages. Here, we describe an unusual snoRNA (TtnuCD32) belonging to the box C/D subgroup from the ciliate Tetrahymena thermophila. We show that TtnuCD32 is unlikely to function as a modification guide snoRNA and that it is critical for cell viability. Cell lines with genetic knock-down of TtnuCD32 were impaired in growth and displayed two novel and apparently unrelated phenotypes. The most prominent phenotype is the accumulation of processing intermediates of 5.8S rRNA. The second phenotype is the decrease in abundance of a ~100 nt 26S rRNA fragment of unknown function. Sequence analysis demonstrated that TtnuCD32 share features with the essential snoRNA U14 but an alternative candidate (TtnuCD25) was more closely related to other U14 sequences. This, together with the fact that the observed rRNA processing phenotypes were not similar to what has been observed in U14 depleted cells, suggests that TtnuCD32 is a U14 homolog that has gained novel functions.


Subject(s)
Gene Knockdown Techniques , RNA Processing, Post-Transcriptional/genetics , RNA, Ribosomal, 5.8S/genetics , RNA, Ribosomal/genetics , RNA, Small Nucleolar/genetics , Tetrahymena/genetics , Base Sequence , Cell Survival , Conserved Sequence , Gene Expression Regulation , Genome , Methylation , Nucleic Acid Conformation , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , RNA, Guide, Kinetoplastida/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal, 5.8S/chemistry , RNA, Small Nucleolar/chemistry
8.
Molecules ; 21(11)2016 Oct 31.
Article in English | MEDLINE | ID: mdl-27809244

ABSTRACT

Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.


Subject(s)
Heat-Shock Response/physiology , Introns , Myxomycetes/metabolism , RNA, Catalytic/biosynthesis , Myxomycetes/genetics , RNA, Catalytic/genetics
9.
Nucleic Acids Res ; 40(3): 1267-81, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21967850

ABSTRACT

The ciliate Tetrahymena thermophila is an important eukaryotic model organism that has been used in pioneering studies of general phenomena, such as ribozymes, telomeres, chromatin structure and genome reorganization. Recent work has shown that Tetrahymena has many classes of small RNA molecules expressed during vegetative growth or sexual reorganization. In order to get an overview of medium-sized (40-500 nt) RNAs expressed from the Tetrahymena genome, we created a size-fractionated cDNA library from macronuclear RNA and analyzed 80 RNAs, most of which were previously unknown. The most abundant class was small nucleolar RNAs (snoRNAs), many of which are formed by an unusual maturation pathway. The modifications guided by the snoRNAs were analyzed bioinformatically and experimentally and many Tetrahymena-specific modifications were found, including several in an essential, but not conserved domain of ribosomal RNA. Of particular interest, we detected two methylations in the 5'-end of U6 small nuclear RNA (snRNA) that has an unusual structure in Tetrahymena. Further, we found a candidate for the first U8 outside metazoans, and an unusual U14 candidate. In addition, a number of candidates for new non-coding RNAs were characterized by expression analysis at different growth conditions.


Subject(s)
Macronucleus/genetics , RNA, Protozoan/chemistry , RNA, Untranslated/chemistry , Tetrahymena thermophila/genetics , Base Sequence , Cells, Cultured , Conserved Sequence , Gene Library , Genome, Protozoan , Methylation , Molecular Sequence Data , Pseudouridine/metabolism , RNA, Protozoan/classification , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/metabolism , RNA, Small Nucleolar/chemistry , RNA, Small Nucleolar/classification , RNA, Untranslated/classification , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Tetrahymena thermophila/metabolism
10.
Mol Biol Cell ; 23(1): 36-44, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22049026

ABSTRACT

RNase T2 enzymes are produced by a wide range of organisms and have been implicated to function in diverse cellular processes, including stress-induced anticodon loop cleavage of mature tRNAs to generate tRNA halves. Here we describe a family of eight RNase T2 genes (RNT2A-RNT2H) in the ciliate Tetrahymena thermophila. We constructed strains lacking individual or combinations of these RNT2 genes that were viable but had distinct cellular and molecular phenotypes. In strains lacking only one Rnt2 protein or lacking a subfamily of three catalytically inactive Rnt2 proteins, starvation-induced tRNA fragments continued to accumulate, with only a minor change in fragment profile in one strain. We therefore generated strains lacking pairwise combinations of the top three candidates for Rnt2 tRNases. Each of these strains showed a distinct starvation-specific profile of tRNA and rRNA fragment accumulation. These results, the delineation of a broadened range of conditions that induce the accumulation of tRNA halves, and the demonstration of a predominantly ribonucleoprotein-free state of tRNA halves in cell extract suggest that ciliate tRNA halves are degradation intermediates in an autophagy pathway induced by growth arrest that functions to recycle idle protein synthesis machinery.


Subject(s)
Endoribonucleases/metabolism , Protozoan Proteins/metabolism , RNA, Protozoan/metabolism , RNA, Ribosomal/metabolism , RNA, Transfer/metabolism , Tetrahymena thermophila/enzymology , Amino Acid Sequence , Autophagy , Catalytic Domain , Cell Cycle Checkpoints , Conserved Sequence , Endoribonucleases/chemistry , Endoribonucleases/genetics , Gene Knockout Techniques , Molecular Sequence Data , Phenotype , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Stress, Physiological , Tetrahymena thermophila/genetics , Tetrahymena thermophila/physiology
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