John LaCava

John LaCava
University of Groningen | RUG · European Research Institute for the Biology of Ageing

PhD

About

49
Publications
24,846
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
2,405
Citations

Publications

Publications (49)
Article
Full-text available
A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely und...
Article
Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutati...
Preprint
Full-text available
A loss of the checkpoint kinase ATM leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and a high risk to cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to wh...
Article
Full-text available
Background Most patients with systemic lupus erythematosus (SLE) have IgG autoantibodies against the RNA-binding p40 (ORF1p) protein encoded by the L1 retroelement. This study tested if these autoantibodies are also present in children with pediatric SLE (pSLE) and if the p40 protein itself could be detected in immune cells. Methods Autoantibodies...
Article
Full-text available
A 5',7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). Interest in the CBC has recently renewed due to its participation in RN...
Article
Full-text available
The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5'cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein...
Preprint
Full-text available
The nuclear Cap Binding Complex (CBC), consisting of Nuclear Cap Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5'cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein...
Preprint
Retrotransposons are genomic DNA sequences that are capable of copying themselves to new genomic locations via RNA intermediates; LINE-1 is the only retrotransposon that remains autonomous and active in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues, but LINE-1 is active in many cancers. Recent studies using LINE-1...
Preprint
Full-text available
A 5', 7-methylguanosine cap is a quintessential feature of RNA polymerase II-transcribed RNAs, and a textbook aspect of co-transcriptional RNA processing. The cap is bound by the cap-binding complex (CBC), canonically consisting of nuclear cap-binding proteins 1 and 2 (NCBP1/2). The CBC has come under renewed investigative interest in recent years...
Article
A Think-Tank Meeting was convened by the National Cancer Institute (NCI) to solicit experts’ opinion on the development and application of multi-omic single-cell analyses, and especially single-cell proteomics, to improve the development of a new generation of biomarkers for cancer risk, early detection, diagnosis, prognosis as well as to discuss t...
Chapter
In humans, the RNA exosome consists of an enzymatically inactive nine-subunit core, with ribonucleolytic activity contributed by additional components. Several cofactor complexes also interact with the exosome—these enable the recruitment of, and specify the activity upon, diverse substrates. Affinity capture coupled with mass spectrometry has prov...
Book
This volume provides a cross-section of RNA exosome research protocols, applied to a diversity of model organisms. Chapters guide readers through methods that e.g. delineate eukaryotic exosomes’ origins in prokaryotes, probe its RNA substrates, adapter complexes, and macromolecular interaction of networks, and establish critical structure-function...
Article
Full-text available
Background: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF...
Article
We are delighted that our discovery of autoantibodies directed against LINE‐1 retroelement‐encoded p40/ORF1p in SLE patients (1) has been so rapidly replicated and we thank Dr. Crow for her excellent summary of our findings. Her group's detection of LINE‐1 mRNA and immunoreactive p40 protein in salivary gland biopsies from primary Sjögren's syndrom...
Preprint
Full-text available
Proteins are the chief effectors of cell biology and their functions are typically carried out in the context of multi-protein assemblies; large collections of such interacting protein assemblies are often referred to as interactomes. Knowing the constituents of protein complexes is therefore important for investigating their molecular biology. Man...
Preprint
Full-text available
A bstract Background Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcriptio...
Article
Objective: The long interspersed nuclear element 1 (LINE-1) encodes two proteins, the RNA-binding p40 and the endonuclease and reverse transcriptase (ORF2p); both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with the LINE-1 RNA and additional RNA-binding proteins, some of which are well-known autoantig...
Article
Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a 'copy-and-paste' fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides lon...
Article
Mutations in the ER chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We used mass spectrometry proteomics to identify CALR-mutan...
Article
Full-text available
Polycomb repressive complex 1 (PRC1) plays essential roles in cell fate decisions and development. However, its role in cancer is less well understood. Here, we show that RNF2, encoding RING1B, and canonical PRC1 (cPRC1) genes are overexpressed in breast cancer. We find that cPRC1 complexes functionally associate with ERα and its pioneer factor FOX...
Article
GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate...
Article
Full-text available
LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1's...
Article
Full-text available
Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mas...
Article
Full-text available
Nuclear RNA metabolism is influenced by protein complexes connecting to both RNA-productive and -destructive pathways. The ZC3H18 protein binds the cap-binding complex (CBC), universally present on capped RNAs, while also associating with the nuclear exosome targeting (NEXT) complex, linking to RNA decay. To dissect ZC3H18 function, we conducted in...
Article
Full-text available
The SEA/GATOR complex is an essential regulator of the mTORC1 pathway. In mammals the GATOR1 complex is composed of the proteins DEPDC5, NPRL2 and NPRL3. GATOR1 serves as an mTORC1 inhibitor and activates the mTORC1-modulating RagA GTPase. However, several GATOR members have mTORC1 independent functions. Here we characterize mammalian cells overexp...
Article
Full-text available
Polycomb group proteins (PcG) are transcriptional repressors that control cell identity and development. In mammals, five different CBX proteins associate with the core Polycomb repressive complex 1 (PRC1). In mouse embryonic stem cells (ESCs), CBX6 and CBX7 are the most highly expressed CBX family members. CBX7 has been recently characterized, but...
Preprint
Full-text available
1. Summary Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications and q...
Preprint
Full-text available
LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology to aid its proliferation. However, a cle...
Article
The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from HEK-293 cell...
Article
The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3'-5' exoribonuclease. The follo...
Article
Full-text available
Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affi...
Article
Full-text available
As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces...
Article
This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation w...
Article
Full-text available
Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacryl...
Article
Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, perm...
Article
This protocol describes the isolation of native protein complexes by density gradient ultracentrifugation. The outcome of an affinity capture and native elution experiment is generally a mixture of (1) the complex(es) associated with the protein of interest under the specific conditions of capture, (2) fragments of the complex generated by degradat...
Article
Full-text available
The LINE-1 retrotransposon (L1) encodes two proteins, ORF1p and ORF2p, which bind to the L1 RNA in cis, forming a ribonucleoprotein (RNP) complex that is critical for retrotransposition. Interactions with both permissive and repressive host factors pervade every step of the L1 life cycle. Until recently, limitations in detection and production prec...
Article
We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affe...
Article
Full-text available
Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell...
Article
Full-text available
LINE-1 (L1) elements are endogenous retrotransposons active in mammalian genomes. The L1 RNA is bicistronic, encoding two non-overlapping open reading frames, ORF1 and ORF2, whose protein products (ORF1p and ORF2p) bind the L1 RNA to form a ribonucleoprotein (RNP) complex that is presumed to be a critical retrotransposition intermediate. However, O...
Article
Full-text available
Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associat...
Article
LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock...
Article
Here we report a modified peptide reagent useful for the rapid, native elution of protein complexes containing a Protein-A-tagged component. We tested this reagent for the elution of tagged endogenous protein complexes from yeast (Nup53p/Nup170p dimer; Nup1p/Kap95p/Kap60p trimer; pentameric GINS complex) and bacteria (RNAPII holoenzyme). The majori...
Article
Full-text available
An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous-level tagged proteins. Isolations of triple-FLAG and...
Article
The exosome complex of 3'-->5' exonucleases is an important component of the RNA-processing machinery in eukaryotes. This complex functions in the accurate processing of nuclear RNA precursors and in the degradation of RNAs in both the nucleus and the cytoplasm. However, it has been unclear how different classes of substrate are distinguished from...
Article
The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a know...

Questions

Questions (10)
Question
So - we are occasionally using methanol/ammonium acetate precipitation of proteins - from phenol or aqueous solution. Essentially as described in -
Faurobert, M., Pelpoir, E. & Chaïb, J. Phenol Extraction of Proteins for Proteomic Studies of Recalcitrant Plant Tissues. in Plant Proteomics: Methods and Protocols (eds. Thiellement, H., Zivy, M, Damerval, C & Méchin, V.) vol. 355 9–14 (2007).
This chapter references - Hurkman, W. J. & Tanaka, C. K. Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis. Plant Physiol 81, 802–806 (1986).
And you can find e.g. Wu, X., Xiong, E., Wang, W., Scali, M. & Cresti, M. Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis. Nat Protoc 9, 362–374 (2014).
And other references to this approach can be found.
Now, what I am missing (and maybe I am going nuts and overlooked it in these papers, somehow) is the significance of ammonium acetate here - it is a natural pH 7 buffer (at least in water) and pH control is a good thing in most cases - and I believe I read somewhere (but can't find where now) that alcohol (organic?) precipitations of proteins are most efficient/reproducible at near neutral pH - is this an isoelectric point consideration for an 'average' protein (in which case I thought slightly acidic would be better based on 'average' pI [disclaimer - in aqueous solution]).
Then I came across this:
Nickerson, J. L. & Doucette, A. A. Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry. J Proteome Res 19, 2035–2042 (2020).
And it made me consider if the contribution of the ammonium acetate is the ionic strength it imparts to the solution (or, ionic strength + neutral pH buffering together).
I am still on the hunt for the reference I believe I read long ago that addressed this matter -- but in the mean time I figured I would solicit feedback from the community to perhaps expedite me arrival back at the correct answer.
Question
I am trying to troubleshoot something I noticed recently - we are accustomed to assaying RNA-dependent interactions in immunoprecipitated fractions by treatment with RNases / nucleases (e.g. RNase A/T1, RNAse H, benzonase etc).
Although we do release proteins from the beads on account of the RNase lability - we found two interesting things happen:
(1) the target protein can no longer be natively eluted from the beads
(2) if we conduct a second IP on the released proteins - those can also not be natively released.
My take on this is that removal of RNA from RNA binding proteins makes them irreversibly bind to the bead surface and that is what I am currently investigating by several strategies - but I would like to get feedback from the community if anyone else has observed and/or already solved this problem. Happy to share the tricks I am trying too.
Cheers
John
Question
Can anyone lend an opinion vis a vis RNA QC using:
Agilent 2100 Bioanalyzer RIN
Invitrogen Qubit RNA IQ
I have traditionally used RIN and I am thinking of moving over to IQ - what are the caveats and provisos? Is IQ a valid substitute for (or, perhaps even better than) RIN?
Let me know your thoughts and opinions.
Question
Greetings,
So, we're developing some hybrid protocols that include traditional TRIzol/organic extractions, phase lock tubes, and also column purifications of RNA.
I noticed that in the modern TRIzol protocols you sometimes see "DO NOT VORTEX" warnings, or if not, still the procedure calls for pipette mixing instead of vortexing (and may not even mention mixing when chloroform is added?!).
This seems silly to me. My understanding is that this can come from two possible places:
(1) this is carry over from DNA phenol/chroloform extraction protocols where intense vortexing can shear large chromatin fragments (however, this proviso is not relevant to an RNA extraction procedure, IMO).
(2) this is related to the assumption that people don't know how to vortex properly and that it is technically possible (albeit, IMO, extraordinarily rare to the point where you need to be trying to screw up your experiment) to vortex and yet not manage to properly mix the chloroform and aqueous phases IF the chloroform remains at the bottom of the tube during vortexing. Which, OK, I can understand a warning to be sure to vortex properly, or mix properly by other means.
I have also noticed prescribed periods of (seemingly static) incubation to dissociate RNPs in TRIzol... has any one experienced that e.g. 1 min or vortexing in the presence of TRIzol would not be enough to achieve "full denaturation" of target RNPs? I guess the risk is that such partly denatured material may partition to the interphase.
Full disclosure - I am extracting RNA from IPs not from whole cell extracts, so, my denaturation demands may be more easily fulfilled. But in any case, extended static incubation just seems... odd to me. I am open to be convinced otherwise.
Lastly, I also noticed these DO NOT VORTEX warnings in Phase Lock protocols - e.g.
My question is - is this an issue where vortexing adversely affects Phase Lock? I presume this is entirely possible... or is this an extension/retention of lore in points 1 and 2 above?
My approach would be... add TRIzol, vortex 1 min, transfer to new tube, add chloroform, vortex 1min, transfer to phase lock.... spin.
By comparison currently, we are adding TRIzol, vortex 1 min, transfer to Phase Lock, incubating 2 min, adding chloroform, mixing vigorously by hand 15 sec (a la manufacturer's protocol), then centrifuging.
OK, so, what we are doing is only about a min longer than what I would do -- and involves one less change of tubes (a good thing)... but nevertheless I want to get to the bottom of the DO NOT VORTEX warnings as they apply generally to TRIzol (I don't think it applies, actually) and specifically to Phase Lock (I think it may apply - but I don't have explicit expression of the reason - I suspect the gel/grease may not work as well if dispersed by vortexing).
All opinions welcomed. Including those related to the length of time it takes to fully denature RNPs in TRIzol.
Cheers
J
Question
We're using filter tips for some delicate operations - but when we need to take phases (such as during Trizol extraction), it is difficult to see the position of the tip because the tips are clear / natural in color. I'd like to get the "traditional" yellow 200 ul and blue 1000 ul tips but also with filters -- I don't find them easily on a web search. Can anyone recommend?
Cheers
John
Question
I am interested in the RNase inhibitor referred to as pdUppAp, described in the paper:
Potent Inhibition of Mammalian Ribonucleases by 3􏰀,5􏰀-Pyrophosphate-linked Nucleotides
Nello Russo and Robert Shapiro
THE JOURNAL OF BIOLOGICAL CHEMISTRY
1999
A quick search did not seem to turn this up on the commercial market - so, figured I see if anyone here is aware of it being available - or if it does by some other trade name.
Also, I noted this paper:
Anyone with experience with that, I'd be interested in a chat.

Network

Cited By

Projects

Projects (2)
Archived project
Affinity-based interactome analyses and in vitro enzymatic assays are being used to characterize L1 intermediates partitioning between the nucleus and cytoplasm of the cell.