The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Expression of the recombinant human U1-70K protein in COS cells resulted in its rapid transport to the nucleus, even when binding to U1 RNA was debilitated. Deletion analysis of the U1-70K protein revealed the existence of two segments of the protein which were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein which was capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif NLS demonstrated that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus.

Download Full-text

The Human But Not theXenopusRNA-editing Enzyme ADAR1 Has an Atypical Nuclear Localization Signal and Displays the Characteristics of a Shuttling Protein

Molecular Biology of the Cell ◽

10.1091/mbc.12.7.1911 ◽

2001 ◽

Vol 12 (7) ◽

pp. 1911-1924 ◽

Cited By ~ 69

Author(s):

Christian R. Eckmann ◽

Andrea Neunteufl ◽

Lydia Pfaffstetter ◽

Michael F. Jantsch

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Rna Binding ◽

Binding Domain ◽

Biologically Active ◽

Nuclear Accumulation ◽

Dual Function ◽

Double Stranded Rna ◽

Rna Binding Domain ◽

Editing Enzyme

The RNA-editing enzyme ADAR1 (adenosine deaminase that acts on RNA) is a bona fide nuclear enzyme that has been cloned from several vertebrate species. Putative nuclear localization signals (NLSs) have been identified in the aminoterminal regions of both human andXenopus ADAR1. Here we show that neither of these predicted NLSs is biologically active. Instead, we could identify a short basic region located upstream of the RNA-binding domains ofXenopus ADAR1 to be necessary and sufficient for nuclear import. In contrast, the homologous region in human ADAR1 does not display NLS activity. Instead, we could map an NLS in human ADAR1 that overlaps with its third double-stranded RNA-binding domain. Interestingly, the NLS activity displayed by this double-stranded RNA-binding domain does not depend on RNA binding, therefore showing a dual function for this domain. Furthermore, nuclear accumulation of human (hs) ADAR1 is transcription dependent and can be stimulated by LMB, an inhibitor of Crm1-dependent nuclear export, indicating that hsADAR1 can move between the nucleus and cytoplasm. Regulated nuclear import and export of hsADAR1 can provide an excellent mechanism to control nuclear concentration of this editing enzyme thereby preventing hyperediting of structured nuclear RNAs.

Download Full-text

The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4662-4670.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4662-4670

Author(s):

J M Romac ◽

D H Graff ◽

J D Keene

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Rna Binding ◽

Binding Domain ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Recognition Motif ◽

Small Nuclear Ribonucleoprotein ◽

Localization Signal ◽

Nuclear Ribonucleoprotein

Expression of the recombinant human U1-70K protein in COS cells resulted in its rapid transport to the nucleus, even when binding to U1 RNA was debilitated. Deletion analysis of the U1-70K protein revealed the existence of two segments of the protein which were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein which was capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif NLS demonstrated that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus.

Download Full-text

Straightening of bulged RNA by the double-stranded RNA-binding domain from the protein kinase PKR

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.011355798 ◽

2000 ◽

Vol 97 (26) ◽

pp. 14162-14167 ◽

Cited By ~ 17

Author(s):

X. Zheng ◽

P. C. Bevilacqua

Keyword(s):

Protein Kinase ◽

Rna Binding ◽

Binding Domain ◽

Double Stranded Rna ◽

Rna Binding Domain

Download Full-text

Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

Biochemical Journal ◽

10.1042/bj20050694 ◽

2005 ◽

Vol 393 (1) ◽

pp. 245-254 ◽

Cited By ~ 32

Author(s):

Catherine Martel ◽

Paolo Macchi ◽

Luc Furic ◽

Michael A. Kiebler ◽

Luc Desgroseillers

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Mammalian Cells ◽

Rna Binding ◽

Binding Activity ◽

Nuclear Trafficking ◽

Binding Domains ◽

Bipartite Nuclear Localization Signal ◽

Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

Download Full-text

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.358 ◽

1995 ◽

Vol 15 (1) ◽

pp. 358-364 ◽

Cited By ~ 61

Author(s):

S R Green ◽

L Manche ◽

M B Mathews

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Rna Binding ◽

Rna Binding Proteins ◽

Alpha Helix ◽

Binding Domain ◽

Single Amino Acid ◽

Double Stranded Rna ◽

C Terminus ◽

Rna Binding Domain

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.

Download Full-text

Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme

Scientific Reports ◽

10.1038/s41598-020-76829-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Richard Izrael ◽

Lívia Marton ◽

Gergely N. Nagy ◽

Hajnalka L. Pálinkás ◽

Nóra Kucsma ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Cell Line ◽

Nuclear Localization ◽

Nuclear Localization Signal ◽

Membrane Binding ◽

Binding Domain ◽

Regulatory Function ◽

Phosphocholine Cytidylyltransferase ◽

Phosphatidylcholine Biosynthesis ◽

Localization Signal

AbstractThe phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

Download Full-text