What is an NLS and what is its purpose?
A nuclear localization signal or sequence (NLS) is an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.
What is NLS in a plasmid?
Nuclear localization signals (NLSs) are known to be involved in the active transport of exogenous proteins and probes into the nucleus. However, stimulation of nuclear localization of plasmid DNA has yet to be confirmed completely.
What is this NLS and why do we need it for use in eukaryotic cells?
Nuclear localization signals (NLSs) allow proteins to be recognized by the importin/karyopherin pathway and internalized into the eukaryotic cell nucleus (1). A number of NLSs in proteins of prokaryotic origin has been reported.
Why would a small protein have NLS?
Why would a small protein have an NLS, when it naturally diffuses across the nuclear pore complex without one? 1. The NLS will be used to actively import the protein into the nucleus, leaving very little of the protein in the cytoplasm.
What proteins have NLS?
Diverse proteins, such as transcription factors, histones, and cell cycle regulators, need to be transported into the nucleus through the NPC after their synthesis, which necessitates the presence of a nuclear localization signal (NLS) on these cargo proteins [9].
What protein does NLS bind to?
Importin α binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin β-dependent import pathway. In humans, only one importin β is known to interact with importin α, while six α importins have been described.
Why is the NLS positively charged?
NLS peptides are positively charged because their sequence is rich in arginines and lysines, and therefore can bind negatively charged pDNA by electrostatic interactions (24).
Why are nuclear localization sequences NLS not removed after nuclear proteins are transported to the nucleus?
Nuclear localization signals are not cleaved off after transport into the nucleus. This is presumably because nuclear proteins need to be imported repeatedly, once after every cell division.
How is nuclear localization measured?
Microscopy vs Biochemical Fractionation. There are two basic ways to assess the nuclear localization of a protein, microscopy and biochemical fractionation.
What is NLS in biology?
Nuclear localization signals (NLS) are conserved amino acid sequences that target proteins for import into the nucleus. From: Case Studies in Cell Biology, 2016.
Is the NLS on the N terminus?
We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK.
Why would a small protein have an NLS when it naturally diffuses across the nuclear pore complex without one?
Why would a small protein have an NLS, when it naturally diffuses across the nuclear pore complex without one? - The NLS will be used to actively import the protein into the nucleus, leaving very little of the protein in the cytoplasm. Diffusion alone would not drive all the protein into the nucleus.
Why is the NLS positively charged?
NLS peptides are positively charged because their sequence is rich in arginines and lysines, and therefore can bind negatively charged pDNA by electrostatic interactions (24).
What property do nuclear localization signal sequences have?
D Nuclear localization signal (NLS) sequences NLS sequences are typically short peptide sequences responsible for direct import of proteins into the nucleus. In general, these sequences contain a high proportion of the basic amino acids lysine and arginine (Lanford and Butel, 1984).
What is nuclear retention?
Nuclear retention of incompletely spliced or mature mRNAs emerges as a novel, previously underappreciated layer of gene regulation, which enables the cell to rapidly respond to stress, viral infection, differentiation cues or changing environmental conditions.
Do all transcription factors have nuclear localization signals?
Most transcription factors contain one or more nuclear localization signal (NLS),1 which, when recognized by nuclear transport proteins, results in the translocation of the transcription factor to the nuclear pore complex. Subsequent translocation across the nuclear membrane occurs in an ATP-dependent fashion (1).
What is SV40 in biology?
SV40. SV40 is an abbreviation for simian vacuolating virus 40 or simian virus 40, a polyomavirus that is found in both monkeys and humans. Like other polyomaviruses, SV40 is a DNA virus that has the potential to cause tumors in animals, but most often persists as a latent infection. SV40 has been widely studied as a model eukaryotic virus, ...
What is SV40 in animals?
SV40 is an abbreviation for simian vacuolating virus 40 or simian virus 40, a polyomavirus that is found in both monkeys and humans. Like other polyomaviruses, SV40 is a DNA virus that has the potential to cause tumors in animals, but most often persists as a latent infection. SV40 has been widely studied as a model eukaryotic virus, ...
What is SV40 in monkeys?
However, in monkeys that are immunodeficient —due to, for example, infection with simian immunodeficiency virus —SV40 acts much like the human JC and BK polyomaviruses, producing kidney disease and sometimes a demyelinating disease similar to progressive multifocal leukoencephalopathy.
When was SV40 first identified?
SV40 was first identified by Ben Sweet and Maurice Hilleman in 1960 when they found that between 10-30% of polio vaccines in the USA were contaminated with SV40. In 1962, Bernice Eddy described the SV40 oncogenic function inducing sarcoma and ependymomas in hamsters inoculated with monkeys cells infected with SV40. The complete viral genome was sequenced by Fiers and his team at the University of Ghent ( Belgium) in 1978.
Is SV40 harmful to humans?
This 2004 announcement is in contrast to a 2002 study performed by The National Academy of Sciences Immunization Safety Review committee that stated, "The committee concludes that the biological evidence is moderate that SV40 exposure could lead to cancer in humans under natural conditions.”.
Does SV40 cause cancer?
The hypothesis that SV40 might cause cancer in humans has been a particularly controversial area of research. It is currently unclear whether SV40 has any role in causing tumors. As a result of these uncertainties, academic opinion remains divided, with some arguing that this hypothesis is not supported by the data and others arguing that some cancers may involve SV40. The US National Cancer Institute announced in 2004 that although SV40 does cause cancer in some animal models, "substantial epidemiological evidence has accumulated to indicate that SV40 likely does not cause cancer in humans". This announcement was based on two studies. This 2004 announcement is in contrast to a 2002 study performed by The National Academy of Sciences Immunization Safety Review committee that stated, "The committee concludes that the biological evidence is moderate that SV40 exposure could lead to cancer in humans under natural conditions.”
Is SV40 a co-carcinogen?
SV40 may act as a co-carcinogen with crocidolite asbestos to cause mesothelioma. The mechanism may involve suppression of the transcriptional properties of tumor suppressor p53 in humans by the SV40 large T antigen and SV40 small T-antigen. Tumor suppressor p53 is responsible for initiating regulated cell death (" apoptosis "), ...
What is NLS used for?
In recent years, NLS were widely used in cancer treatment and viral infection prevention [ 13, 14, 15 ], and researchers paid more attention to identifying novel NLS motifs and the import nucleoporins that recognize and bind them. Here, we categorized NLS according to their residue composition into classical (cNLS), non-classical (ncNLS), and other types.
What is the role of NLS in the nucleus?
The NLS is recognized by the corresponding nuclear transporters, which can interact with nucleoporins to help NLS-containing proteins reach the nucleus through NPCs [ 10 ]. Due to the complex roles of nuclear proteins, NLS-mediated nuclear transport is a highly regulated process.
What are the two categories of cNLs?
As shown in Table 1, the cNLS encompass two categories, termed “monopartite” (MP) and “bipartite” (BP) [ 16 ]. MP NLS are a single cluster composed of 4–8 basic amino acids, which generally contains 4 or more positively charged residues, that is, arginine (R) or lysine (K). The characteristic motif of MP NLS is usually defined as K (K/R) X (K/R), where X can be any residue [ 17 ]. For example, the NLS of SV40 large T-antigen is 126 PKKKRKV 132, with five consecutive positively charged amino acids (KKKRK). Interestingly, studies have shown that its reverse sequence has no nuclear transport function [ 12 ]. Furthermore, if the third amino acid lysine (K) of SV40 large T antigen NLS is mutated to threonine (T), its nuclear transport function is also lost [ 18 ].
Which proteins do not have NLS?
One example is a specific armadillo repeats of the β-catenin protein, a key mediator of Wnt signaling [ 70 ]. The β-catenin has three distinct transport sequences: the N-terminal tail, C-terminal tail and Armadillo repeats10-12 [ 71 ]. These three regions, despite sharing no apparent sequence homology, are capable of binding Nup358, Nup62, Nup98 and Nup153 of the NPC, and thus of directly mediating β-catenin entry to the nucleus via sequential and transient interactions [ 72 ]. Additionally, several publications showed that it is the direct interaction of importin β11 (IPO11) with β-catenin which mediates its nuclear import [ 73 ].
What is the function of NLS?
From the name, the main function of NLS is inside-nucleus signaling through interactions with importin in the nuclear pore complex. The positive charge of NLS allows it alone or with other peptides to be utilized for the delivery of QDs in cells and their nuclei. Chen and Gerion accomplished the delivery of CdSe/ZnS QDs conjugated with NLS derived from SV40 T antigen in cells and their nuclei.36 On the other hand, only inside-cell not inside-nucleus delivery was detected for QDs conjugated with a random peptide sequence. Similarly, Rozenzhak et al. utilized a carrier peptide (Pep-1) for the delivery of QD–streptavidin, QD–NLS, and QD–GH3 domain sequence (derived from the Grim protein) conjugates in cells. 81 They found that Pep-1 serves only as a transmembrane carrier of QDs. The complex between bioconjugated QDs and Pep-1 dissociated in the cytoplasm and resulted in the release of QD–streptavidin, QD–NLS, and QD–GH3. Thus, QD–NLS and QD–GH3 conjugates delivered in the cytoplasm further target nucleus and mitochondria, respectively. Hoshino et al. utilized NLS for the delivery of CdSe/ZnS QD–eGFP conjugates in HEK293T cells. 82 They successfully achieved delivery of QD–eGFP conjugates for the expression of the GFP gene in the cells.
What is NLS in RNA?
Nuclear localization signals (NLS) are reported for the CPsV, MiLBVV and RWMV polymerases and for the RNA-2-encoded proteins of CPsV and MiLBVV.
What are NLS sequences?
NLS sequences are typically short peptide sequences responsible for direct import of proteins into the nucleus. In general, these sequences contain a high proportion of the basic amino acids lysine and arginine ( Lanford and Butel, 1984). Frequently, amino acids such as proline, which disrupt helical domains, are also present. NLS sequences are frequently present within viral proteins, or, as in the case of adenovirus, the nuclear localizing proteins are covalently linked to the 5’ end of the DNA at the terminal dCMP. A variety of viral nucleic acids (HIV-2, influenza virus, SV40, and adenovirus) are guided through the nuclear pore complex with assistance from at least one NLS-containing viral protein. The best characterized NLS is that present in 92-kDa SV40 large T-antigen. Cytoplasmic microinjection of a plasmid complexed with Cys-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-amide has been shown to give enhanced gene expression (Fraley et al., 1980). However, mutations of any lysine residue of SV40 large T-antigen are reported to abolish nuclear accumulation. The minimal sequence that directed a pyruvate kinase fusion protein to the nucleus was Pro-Lys-Lys-Lys-Arg-Lys-Val. Synthetic peptides containing this sequence also targeted cross-linked carrier proteins to the nucleus. Unlike SV40 large T-antigen, the NLS sequence of influenza virus nucleoprotein, Ala-Ala-Phe-Glu-Asp-Leu-Arg-Val-Leu-Ser, has only one basic residue.
Is there a consensus sequence for NLS?
Although there is no single consensus sequence for NLSs, they possess several common features. Typically, an NLS is rich in basic amino acids, not cleaved from the protein after import, and functionally independent of its position within the protein molecule.
Is NLS present in the DBD?
No NLS activity was present in the DBD, which in most nuclear receptors is highly conserved in structure and sequence; at least 40% of the sequence is identical among all nuclear receptors (Chen & Young, 2010 ). There also was no NLS activity present in the LBD, the domain which harbors NES activity (see Section 4.1 ).
What are the different types of NLS?
There are many other types of NLS, such as the acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcription repressor Matα2, and the complex signals of U snRNPs. Most of these NLSs appear to be recognized directly by specific receptors of the importin β family without the intervention of an importin α-like protein.
What is the NLS signal?
A nuclear localization signal or sequence ( NLS) is an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.
What is bipartite NLS?
The bipartite NLS is now known to represent the major class of NLS found in cellular nuclear proteins and structural analysis has revealed how the signal is recognized by a receptor ( importin α) protein (the structural basis of some monopartite NLSs is also known ).
What are the differences between monopartite and bipartite NLSs?
The major structural differences between the two are that the two basic amino acid clusters in bipartite NLSs are separated by a relatively short spacer sequence (hence bipartite - 2 parts), while monopartite NLSs are not. The first NLS to be discovered was the sequence PKKKRKV in the SV40 Large T-antigen (a monopartite NLS). The NLS of nucleoplasmin, KR [PAATKKAGQA]KKKK, is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. Both signals are recognized by importin α. Importin α contains a bipartite NLS itself, which is specifically recognized by importin β. The latter can be considered the actual import mediator.
Is SV40 a functional NLS?
However, a functional NLS could not be identified in another nuclear protein simply on the basis of similarity to the SV40 NLS.
Is SV40 a bipartite NLS?
One of these elements was similar to the SV40 NLS but was not able to direct a protein to the cell nucleus when attached to a non-nuclear reporter protein. Both elements are required. This kind of NLS has become known as a bipartite classical NLS.
Popular Answers (1)
I would prefer c-myc-NLS (PAAKRVKLD) over SV40 with C terminal tagging. In my own experience, c-myc NLS works better compared to other NLS sequences.
All Answers (10)
I have tried fuse this NLS to the N-terminus or in the middle of a protein. Both works in plants.
Overview
SV40 is an abbreviation for simian vacuolating virus 40 or simian virus 40, a polyomavirus that is found in both monkeys and humans. Like other polyomaviruses, SV40 is a DNA virus that has the potential to cause tumors in animals, but most often persists as a latent infection. SV40 has been widely studied as a model eukaryotic virus, leading to many early discoveries in eukaryotic DNA replication and transcription.
Human disease
The hypothesis that SV40 might cause cancer in humans has been a particularly controversial area of research. It is currently unclear whether SV40 has any role in causing tumors. As a result of these uncertainties, academic opinion remains divided, with some arguing that this hypothesis is not supported by the data and others arguing that some cancers may involve SV40. The US National Cancer Institute announced in 2004 that although SV40 does cause cancer in some ani…
Virology
SV40 consists of an unenveloped icosahedral virion with a closed circular double-stranded DNA genome of 5.2 kb. The virion adheres to cell surface receptors of MHC class I by the virion glycoprotein VP1. Penetration into the cell is through a caveolin vesicle. Inside the cell nucleus, the cellular RNA polymerase II acts to promote early gene expression. This results in an mRNA that is spliced into two segments. The small and large T antigens result from this. The large T antigen h…
Multiplicity reactivation
SV40 is capable of multiplicity reactivation (MR). MR is the process by which two or more virus genomes containing otherwise lethal damage interact within an infected cell to form a viable virus genome. Yamamato and Shimojo observed MR when SV40 virions were irradiated with UV light and allowed to undergo multiple infection of host cells. Hall studied MR when SV 40 virions were exposed to the DNA crosslinking agent 4, 5', 8-trimethylpsoralen. Under conditions in which only …
Transcription
The early promoter for SV40 contains three elements. The TATA box is located approximately 20 base-pairs upstream from the transcriptional start site. The 21 base-pair repeats contain six GC boxes and are the site that determines the direction of transcription. Also, the 72 base-pair repeats are transcriptional enhancers. When the SP1 protein interacts with the 21 base-pair repeats, it binds either the first or the last three GC boxes. Binding the first three initiates early expression, bindin…
Other animals
SV40 is dormant and is asymptomatic in rhesus monkeys. The virus has been found in many macaque populations in the wild, where it rarely causes disease. However, in monkeys that are immunodeficient—due to, for example, infection with simian immunodeficiency virus—SV40 acts much like the human JC and BK polyomaviruses, producing kidney disease and sometimes a demyelinating disease similar to progressive multifocal leukoencephalopathy. In other species, p…
History
SV40 was first identified by Ben Sweet and Maurice Hilleman in 1960 when they found that between 10 and 30% of polio vaccines in the USA were contaminated with SV40. In 1962, Bernice Eddy described the SV40 oncogenic function inducing sarcoma and ependymomas in hamsters inoculated with monkeys cells infected with SV40. The complete viral genome was sequenced by Weissman at Yale University (USA) in 1978 and also by Fiers and his team at the University of Ghent (
See also
• Mason-Pfizer monkey virus packaging signal
• SV40 Cancer Foundation