Biomedical subjects

Kirk M Brown

Publications and source records attributed to Kirk M Brown.

7 recordsLinked to original sources

PubMed2005

Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition.

At least half of all human pre-mRNAs are subject to alternative 3' processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3' UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3' processing factor CFI(m) can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI(m) is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI(m) is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI(m) in poly(A) site recognition. The recognition of three distinct sequence elements by CFI(m), CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.

3' Untranslated Regions↗

PubMed2005

Target accessibility dictates the potency of human RISC.

In this report, we examined the effect of increased target site access on activated human RNA-induced silencing complex (RISC(*)) catalysis. Kinetic studies revealed that siRNA-programmed RISC(*) cleaved target RNA with higher efficiencies when target site access was increased. These results provide evidence that target site access is linked to RISC(*) catalysis.

Base Sequence↗

PubMed2005

Specific and potent RNAi in the nucleus of human cells.

RNA interference (RNAi) has become a research tool to control gene expression in various organisms and holds potential as a new therapeutic strategy. The mechanism of small interfering RNA (siRNA)-mediated RNAi involves target mRNA cleavage and destruction in the cytoplasm. We investigated siRNA-mediated induction of RNAi in the nucleus of human cells. Notably, we observed highly efficient knockdown of small nuclear RNA 7SK by siRNA. siRNA- and microRNA-programmed RNA-induced silencing complexes (RISCs) were present in both cytoplasmic and nuclear compartments and specifically cleaved their perfectly matched target RNA with markedly high efficiencies. Our results provide the first evidence that human RISCs programmed with siRNA are present in the nucleus and can knock down target RNA levels. These studies reveal new roles for the RNAi machinery in modulating post-transcriptional gene expression in the nucleus.

Cell Nucleus↗

PubMed2005

Dissecting RNA-interference pathway with small molecules.

RNA interference (RNAi) is a process whereby short-interfering RNAs (siRNA) silence gene expression in a sequence-specific manner. We have screened a chemical library of substituted dihydropteridinones and identified a nontoxic, cell permeable, and reversible inhibitor of the RNAi pathway in human cells. Biochemical and fluorescence resonance-energy transfer experiments demonstrated that one of the compounds, named ATPA-18, inhibited siRNA unwinding that occurred within 6 hr of siRNA transfection. Extracts prepared from ATPA-18-treated cells also exhibited a decrease in target RNA cleavage by activated RNA-induced silencing complex (RISC*). Interestingly, when activated RISC*, which harbors unwound antisense siRNA, was treated with ATPA-18 in vitro, target RNA cleavage was not affected, indicating that this compound inhibited siRNA unwinding or steps upstream of unwinding in the RNAi pathway. Our results also establish the timing of siRNA unwinding and show that siRNA helicase activity is required for RNAi. ATPA-18 analogs will therefore provide a new class of small molecules for studying RNAi mechanisms in a variety of model organisms and deciphering in vivo genetic functions through reverse genetics.

Cell Line↗

PubMed2003

A mechanism for the regulation of pre-mRNA 3' processing by human cleavage factor Im.

Human cleavage factor I(m) (CFI(m)) is a heterodimeric RNA binding protein complex that functions at an early step in the assembly of the pre-mRNA 3' processing complex. In this report we show that CFI(m) can stimulate both cleavage and poly(A) addition, and can act to suppress poly(A) site cleavage in a sequence-dependent manner. Elevated levels of CFI(m) suppressed cleavage at the primary poly(A) site of the pre-mRNA encoding the 68 kDa subunit of CFI(m). CFI(m)-mediated suppression of poly(A) site cleavage was dependent upon the presence of three copies of an RNA element initially identified by CFI(m)-SELEX. These data provide evidence for a mechanism for the regulation of poly(A) site selection by a basal pre-mRNA 3' processing factor.

3' Untranslated Regions↗

PubMed2002

Preparation and characterization of molecular weight fractions of guar galactomannans using acid and enzymatic hydrolysis.

A procedure is described for the preparation of large amounts of guar galactomannan by acid hydrolysis that yields samples of various molecular weights (MW) with uniform polydispersity. This contrasts with preparation by enzymatic degradation that yields samples with a marked increase in polydispersity and a much broader molecular weight distribution (MWD). Acid hydrolyzed guar samples had a Mark-Houwink-Sakurada (MHS) relationship of [eta]=3.04x10(-4) M(w)(0.747) dl/g and a characteristic ratio of 11.87 as determined by gel permeation chromatography (GPC) and dilute solution viscometry. The Huggins coefficient for degraded guars is much smaller (approximately 0.4) than that of the native guar (approximately 0.79), suggesting a weakening of intermolecular association in guar prepared by acid hydrolysis.

Acids↗

PubMedDate not supplied

Characterization and intermolecular interactions of hydroxypropyl guar solutions.

Aqueous solutions of guar galactomannan and hydroxypropyl guars (HPG) with different molar substitution (MS) levels were studied using dilute solution viscometry and gel permeation chromatography. When guar is modified to HPG, the added hydroxypropyl groups sterically block the hydrogen bonding sites on the guar backbone and reduce the hydrogen bonding attractions between guar molecules. The effects of molar substitution on the intermolecular interactions are inferred from measurements of the Huggins coefficients, which measure intermolecular interactions in dilute solution, and molecular volumes, which reflect intrachain associations. The behavior can be divided into three regimes: (1) at low MS levels (0 < MS < approximately 0.4), there is a sharp decrease in intermolecular interactions as a function of MS; (2) in the intermediate range ( approximately 0.4 < MS < approximately 1.0), interactions become independent of MS; (3) at high substitution levels (MS > approximately 1.0), the temperature dependence of inter- and intramolecular hydrophobic interactions produces a temperature dependence in the Huggins coefficient and molecular volumes that is not seen at lower substitutions. By acid hydrolysis, HPG samples with a range of molecular weights and consistent polydispersities were obtained. On the basis of these samples, the Mark-Houwink-Sakurada parameters and "characteristic ratio" C(infinity) were evaluated for HPG (MS approximately 0.6) and compared to the values for guar. The HPG chain stiffens as the degree of substitution increases.

Galactans↗