Effective expression of small interfering RNA in human cells

2002 ◽  
Vol 20 (5) ◽  
pp. 505-508 ◽  
Author(s):  
Cynthia P. Paul ◽  
Paul D. Good ◽  
Ira Winer ◽  
David R. Engelke
Keyword(s):  
Small Interfering Rna ◽  
Human Cells ◽  
Interfering Rna

scholarly journals NBS1 Knockdown by Small Interfering RNA Increases Ionizing Radiation Mutagenesis and Telomere Association in Human Cells

2005 ◽  
Vol 65 (13) ◽  
pp. 5544-5553 ◽  
Author(s):  
Ying Zhang ◽  
Chang U.K. Lim ◽  
Eli S. Williams ◽  
Junqing Zhou ◽  
Qinming Zhang ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Small Interfering Rna ◽  
Human Cells ◽  
Telomere Association ◽  
Interfering Rna

scholarly journals CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells

2010 ◽  
Vol 30 (6) ◽  
pp. 1486-1494 ◽  
Author(s):  
Xianghua Piao ◽  
Xue Zhang ◽  
Ligang Wu ◽  
Joel G. Belasco
Keyword(s):  
Small Rna ◽  
Small Interfering Rna ◽  
Inhibitory Effect ◽  
Human Cells ◽  
Predominant Role ◽  
Catalytic Subunits ◽  
Present Evidence ◽  
Mrna Targets ◽  
Protein Components ◽  
Interfering Rna

ABSTRACT MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of “off-target” mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.

scholarly journals Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the Integrator complex

2013 ◽  
Vol 24 (18) ◽  
pp. 2954-2965 ◽  
Author(s):  
Jeanne N. Jodoin ◽  
Poojitha Sitaram ◽  
Todd R. Albrecht ◽  
Sarah B. May ◽  
Mohammad Shboul ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Rna Processing ◽  
Small Interfering Rna ◽  
Cytoplasmic Dynein ◽  
Human Cells ◽  
Nuclear Localization Sequence ◽  
Small Nuclear Rnas ◽  
Localization Sequence ◽  
Cultured Human Cells ◽  
Interfering Rna

We previously reported that Asunder (ASUN) is essential for recruitment of dynein motors to the nuclear envelope (NE) and nucleus–centrosome coupling at the onset of cell division in cultured human cells and Drosophila spermatocytes, although the mechanisms underlying this regulation remain unknown. We also identified ASUN as a functional component of Integrator (INT), a multisubunit complex required for 3′-end processing of small nuclear RNAs. We now provide evidence that ASUN acts in the nucleus in concert with other INT components to mediate recruitment of dynein to the NE. Knockdown of other individual INT subunits in HeLa cells recapitulates the loss of perinuclear dynein in ASUN–small interfering RNA cells. Forced localization of ASUN to the cytoplasm via mutation of its nuclear localization sequence blocks its capacity to restore perinuclear dynein in both cultured human cells lacking ASUN and Drosophila asun spermatocytes. In addition, the levels of several INT subunits are reduced at G2/M when dynein is recruited to the NE, suggesting that INT does not directly mediate this step. Taken together, our data support a model in which a nuclear INT complex promotes recruitment of cytoplasmic dynein to the NE, possibly via a mechanism involving RNA processing.

scholarly journals Knockdown of Human Nα-Terminal Acetyltransferase Complex C Leads to p53-Dependent Apoptosis and Aberrant Human Arl8b Localization

2009 ◽  
Vol 29 (13) ◽  
pp. 3569-3581 ◽  
Author(s):  
Kristian K. Starheim ◽  
Darina Gromyko ◽  
Rune Evjenth ◽  
Anita Ryningen ◽  
Jan Erik Varhaug ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Protein Function ◽  
Small Interfering Rna ◽  
Human Cells ◽  
Auxiliary Subunits ◽  
Complex Functions ◽  
Dependent Cell ◽  
Interfering Rna

ABSTRACT Protein Nα-terminal acetylation is one of the most common protein modifications in eukaryotic cells. In yeast, three major complexes, NatA, NatB, and NatC, catalyze nearly all N-terminal acetylation, acetylating specific subsets of protein N termini. In human cells, only the NatA and NatB complexes have been described. We here identify and characterize the human NatC (hNatC) complex, containing the catalytic subunit hMak3 and the auxiliary subunits hMak10 and hMak31. This complex associates with ribosomes, and hMak3 acetylates Met-Leu protein N termini in vitro, suggesting a model in which the human NatC complex functions in cotranslational N-terminal acetylation. Small interfering RNA-mediated knockdown of NatC subunits results in p53-dependent cell death and reduced growth of human cell lines. As a consequence of hMAK3 knockdown, p53 is stabilized and phosphorylated and there is a significant transcriptional activation of proapoptotic genes downstream of p53. Knockdown of hMAK3 alters the subcellular localization of the Arf-like GTPase hArl8b, supporting that hArl8b is a hMak3 substrate in vivo. Taken together, hNatC-mediated N-terminal acetylation is important for maintenance of protein function and cell viability in human cells.

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