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Tariq M Rana

Publications and source records attributed to Tariq M Rana.

At least 19 recordsLinked to original sources
PubMed2007

Illuminating the silence: understanding the structure and function of small RNAs.

RNA interference (RNAi) is triggered by double-stranded RNA helices that have been introduced exogenously into cells as small interfering (si)RNAs or that have been produced endogenously from small non-coding RNAs known as microRNAs (miRNAs). RNAi has become a standard experimental tool and its therapeutic potential is being aggressively harnessed. Understanding the structure and function of small RNAs, such as siRNAs and miRNAs, that trigger RNAi has shed light on the RNAi machinery. In particular, it has highlighted the assembly and function of the RNA-induced silencing complex (RISC), and has provided guidelines to efficiently silence genes for biological research and therapeutic applications of RNAi.

Animals↗
PubMed2006

Discovery of HIV-1 protease inhibitors with picomolar affinities incorporating N-aryl-oxazolidinone-5-carboxamides as novel P2 ligands.

Here, we describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors incorporating N-phenyloxazolidinone-5-carboxamides into the (hydroxyethylamino)sulfonamide scaffold as P2 ligands. Series of inhibitors with variations at the P2 phenyloxazolidinone and the P2' phenylsulfonamide moieties were synthesized. Compounds with the (S)-enantiomer of substituted phenyloxazolidinones at P2 show highly potent inhibitory activities against HIV-1 protease. The inhibitors possessing 3-acetyl, 4-acetyl, and 3-trifluoromethyl groups at the phenyl ring of the oxazolidinone fragment are the most potent in each series, with K(i) values in the low picomolar (pM) range. The electron-donating groups 4-methoxy and 1,3-dioxolane are preferred at P2' phenyl ring, as compounds with other substitutions show lower binding affinities. Attempts to replace the isobutyl group at P1' with small cyclic moieties caused significant loss of affinities in the resulting compounds. Crystal structure analysis of the two most potent inhibitors in complex with the HIV-1 protease provided valuable information on the interactions between the inhibitor and the protease enzyme. In both inhibitor - enzyme complexes, the carbonyl group of the oxazolidinone ring makes hydrogenbond interactions with relatively conserved Asp29 residue of the protease. Potent inhibitors from each series incorporating various phenyloxazolidinone based P2 ligands were selected and their activities against a panel of multidrug-resistant (MDR) protease variants were determined. Interestingly, the most potent protease inhibitor starts out with extremely tight affinity for the wild-type enzyme (K(i) = 0.8 pM), and even against the MDR variants it retains picomolar to low nanomolar K(i), which is highly comparable with the best FDA-approved protease inhibitors.

Amides↗
PubMed2006

Dynamics of nascent mRNA folding and RNA-protein interactions: an alternative TAR RNA structure is involved in the control of HIV-1 mRNA transcription.

HIV-1 Tat protein regulates transcription elongation by binding to the 59 nt TAR RNA stem-loop structure transcribed from the HIV-1 5' long terminal repeat (5'-LTR). This established Tat-TAR interaction was used to investigate mRNA folding and RNA-protein interactions during early transcription elongation from the HIV-1 5'-LTR. Employing a new site-specific photo-cross-linking strategy to isolate transcription elongation complexes at early steps of elongation, we found that Tat interacts with HIV-1 transcripts before the formation of full-length TAR (TAR59). Analysis of RNA secondary structure by free energy profiling and ribonuclease digestion indicated that nascent transcripts folded into an alternative TAR RNA structure (TAR31), which requires only 31 nt to form and includes an analogous Tat-binding bulge structure. Functionally, TAR31, similar to TAR59, acts as a transcriptional terminator in vitro, and mRNA expression from TAR31-deficient HIV-1 5'-LTR mutant promoters is significantly decreased. Our results support a role for TAR31 in the control of HIV-1 mRNA transcription and we propose that this structure is important to stabilize the short early transcripts before the transcription complex commits for processive elongation. Overall, this study demonstrates that RNA folding during HIV-1 transcription is dynamic and that as the nascent RNA chain grows during transcription, it folds into a number of conformations that function to regulate gene expression. Finally, our results provide a new experimental strategy for studying mRNA conformation changes during transcription that can be applied to investigate the folding and function of nascent RNA structures transcribed from other promoters.

Gene Products, tat↗
PubMed2006

Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies.

APOBEC3G is an antiviral host factor capable of inhibiting the replication of both exogenous and endogenous retroviruses as well as hepatitis B, a DNA virus that replicates through an RNA intermediate. To gain insight into the mechanism whereby APOBEC3G restricts retroviral replication, we investigated the subcellular localization of the protein. Herein, we report that APOBEC3G localizes to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of nontranslating mRNAs. Biochemical analysis revealed that APOBEC3G localizes to a ribonucleoprotein complex with other P-body proteins which have established roles in cap-dependent translation (eIF4E and eIF4E-T), translation suppression (RCK/p54), RNA interference-mediated post-transcriptional gene silencing (AGO2), and decapping of mRNA (DCP2). Similar analysis with other APOBEC3 family members revealed a potential link between the localization of APOBEC3G and APOBEC3F to a common ribonucleoprotein complex and P-bodies with potent anti-HIV-1 activity. In addition, we present evidence suggesting that an important role for HIV-1 Vif, which subverts both APOBEC3G and APOBEC3F antiviral function by inducing their degradation, could be to selectively remove these proteins from and/or restrict their localization to P-bodies. Taken together, the results of this study reveal a novel link between innate immunity against retroviruses and P-bodies suggesting that APOBEC3G and APOBEC3F could function in the context of P-bodies to restrict HIV-1 replication.

APOBEC-3G Deaminase↗
PubMed2006

Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54.

RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3' UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3' UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.

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

Mechanism of site-specific psoralen photoadducts formation in triplex DNA directed by psoralen-conjugated oligonucleotides.

Triplex-formation oligonucleotides attached with a photoreactive psoralen molecule (psoTFO) can be used to induce site-specific DNA damage and to control gene expression. Inhibition of transcription by psoralen-cross-linked triplexes results in both arrest and termination of RNA Pol II transcriptional complexes during elongation. To understand the relationship between triplex psoralen cross-linking products and the fate of RNA Pol II elongation complexes, it is important to delineate the mechanism for creating site-specific psoralen photoadducts in a target duplex DNA. To investigate the mechanism of photoadduct-formation by psoralen photo-cross-linking, triplex structures were generated by targeting a DNA duplex with psoTFOs of different lengths. The psoralen photoadducts were then analyzed after UV irradiation, which initiates the psoralen cross-linking reaction. Our results demonstrated that UV irradiation of triplexes formed between a psoTFO and a DNA duplex generated two distinct groups of psoralen photoadducts: monoadducts and psoralen interstrand cross-link products. The formation of these psoralen photoadducts was also photoreversible through exposure to short wavelength UV irradiation. The length of a psoTFO was shown to establish the position at which psoralen was added to the target DNA duplex and determined which photoadducts products formed predominantly. Kinetic experiments that monitored the formation of the psoralen photoadducts also suggested that the length of the psoTFO influenced which photoadducts were preferentially formed at faster rates. Taken together, these studies provide new insight into the mechanism associated with the formation of psoralen photoadducts that are directed by psoTFO during triplex formation.

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↗
PubMed2004

Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5.

BACKGROUND: Several cellular positive and negative elongation factors are involved in regulating RNA polymerase II processivity during transcription elongation in human cells. In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo and in HIV-1 replication has not been completely elucidated. RESULTS: To understand the in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication. CONCLUSIONS: The findings presented here showed that hSpt5 is a bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.

Gene Expression Regulation, Viral↗
PubMed2004

Analysis of HIV-1 viral infectivity factor-mediated proteasome-dependent depletion of APOBEC3G: correlating function and subcellular localization.

To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and nonfunctional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with gamma-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23. When co-expressed, Vif exhibited more pronounced localization to the cytoplasm and reduced the total cellular levels of APOBEC3G but rarely co-localized with APOBEC3G at cytoplasmic bodies. On the contrary, Vif(C114S), which is inactive but continues to interact with APOBEC3G, stably associated with APOBEC3G in the cytoplasm, resulting in complete co-localization at cytoplasmic bodies and a dose-dependent exclusion of Vif(C114S) from the nucleus. Following proteasome inhibition, cytoplasmic APOBEC3G levels increased, and both proteins co-accumulated nonspecifically into a vimentin-encaged aggresome. Furthermore in the presence or absence of APOBEC3G, Vif localization was significantly altered by proteasome inhibition, suggesting that aberrant localization may also contribute to the loss of Vif function. Finally mutations at Vif Ile(9) disrupted the ability of Vif or Vif(C114S) to coimmunoprecipitate and to co-localize with APOBEC3G, suggesting that the N terminus of Vif mediates interactions with APOBEC3G. Taken together, these results demonstrate that cytoplasmic Vif-APOBEC3G interactions are required but are not sufficient for Vif to modulate APOBEC3G and can be monitored by co-localization in vivo.

APOBEC-3G Deaminase↗
PubMed2004

Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells.

RNA interference (RNAi) is the process by which short-interfering RNA (siRNA) target a specific mRNA for degradation through interactions with an RNA-induced silencing complex (RISC). Here, a clear correlation between siRNA localization, cellular uptake, and RNAi activity was discovered by delivering siRNA into cells using siRNA-TAT(47-57) peptide, siRNA-TAT(47-57)-derived oligocarbamate conjugates, or nanoparticles. For successful RNAi, the localization of siRNA was distinctly perinuclear, suggesting that siRNA is targeted to these regions for interactions with RISC to induce RNAi. siRNA sequence variation and the presence of the target mRNA apparently did not change the subcellular localization pattern of siRNA. Intriguingly, siRNA conjugated to TAT(47-57) peptide or TAT(47-57)-derived oligocarbamate resulted in efficient RNAi activity and perinuclear localization of siRNA that was distinctly different from nonconjugated free TAT peptide nucleolar localization. These results suggest that interactions with RISC dictate siRNA localization even when siRNA is conjugated to TAT(47-57) peptide.

Base Sequence↗
PubMed2004

Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1).

The human positive transcription elongation factor P-TEFb is composed of two subunits, cyclin T1 (hCycT1) and CDK9, and is involved in transcriptional regulation of cellular genes as well as human immunodeficiency virus type 1 (HIV-1) mRNA. Replication of HIV-1 requires the Tat protein, which activates elongation of RNA polymerase II at the HIV-1 promoter by interacting with hCycT1. To understand the cellular functions of P-TEFb and to test whether suppression of host proteins such as P-TEFb can modulate HIV infectivity without causing cellular toxicity or lethality, we used RNA interference (RNAi) to specifically knock down P-TEFb expression by degrading hCycT1 or CDK9 mRNA. RNAi-mediated gene silencing of P-TEFb in HeLa cells was not lethal and inhibited Tat transactivation and HIV-1 replication in host cells. We also found that CDK9 protein stability depended on hCycT1 protein levels, suggesting that the formation of P-TEFb CDK-cyclin complexes is required for CDK9 stability. Strikingly, P-TEFb knockdown cells showed normal P-TEFb kinase activity. Our studies suggest the existence of a dynamic equilibrium between active and inactive pools of P-TEFb in the cell and indicate that this equilibrium shifts towards the active kinase form to sustain cell viability when P-TEFb protein levels are reduced. The finding that a P-TEFb knockdown was not lethal and still showed normal P-TEFb kinase activity suggested that there is a critical threshold concentration of activated P-TEFb required for cell viability and HIV replication. These results provide new insights into the regulation of P-TEFb function and suggest the possibility that similar mechanisms for monitoring protein levels to modulate the activity of proteins may exist for the regulation of a variety of other enzymatic pathways.

Cell Line↗
PubMed2003

Selective binding of TAR RNA by a Tat-derived beta-peptide.

[structure: see text] The interaction between the HIV-1 Tat protein and the TAR RNA element in the nascent viral genomic transcript is required for viral replication. An 11-residue beta-peptide (1), an all-beta homologue of the Arg-rich region Tat 47-57, binds TAR RNA with K(d) = 29 +/- 4 nM. A control beta-peptide (2) in which all Arg side chains are replaced by Lys side chains shows increased affinity but decreased specificity for wild-type vs bulge-deleted TAR RNA, as do the alpha-peptide analogues of 1 and 2.

Amino Acid Sequence↗
PubMed2003

Discovery of a small molecule Tat-trans-activation-responsive RNA antagonist that potently inhibits human immunodeficiency virus-1 replication.

Antiretroviral therapy to treat AIDS uses molecules that target the reverse transcriptase and protease enzymes of human immunodeficiency virus, type 1 (HIV-1). A major problem associated with these treatments, however, is the emergence of drug-resistant strains. Thus, there is a compelling need to find drugs against other viral targets. One such target is the interaction between Tat, an HIV-1 regulatory protein essential for viral replication, and trans-activation-responsive (TAR) RNA. Here we describe the design and synthesis of an encoded combinatorial library containing 39,304 unnatural small molecules. Using a rapid high through-put screening technology, we identified 59 compounds. Structure-activity relationship studies led to the synthesis of 19 compounds that bind TAR RNA with high affinities. In the presence of a representative Tat-TAR inhibitor (5 microM TR87), we observed potent and sustained suppression of HIV replication in cultured cells over 24 days. The same concentration of this inhibitor did not exhibit any toxicity in cell cultures or in mice. TR87 was also shown to specifically disrupt Tat-TAR binding in vitro and inhibit Tat-mediated transcriptional activation in vitro and in vivo, providing a strong correlation between its activities and inhibition of HIV-1 replication. These results provide a structural scaffold for further development of new drugs, alone or in combination with other drugs, for treatment of HIV-1-infected individuals. Our results also suggest a general strategy for discovering pharmacophores targeting RNA structures that are essential in progression of other infectious, inflammatory, and genetic diseases.

Animals↗
PubMed2003

Inhibition of HIV tat-TAR interactions by an antisense oligo-2'-O-methylribonucleoside methylphosphonate.

An antisense oligo-2'-O-methylribonucleotide having alternating methylphosphonate/phosphodiester linkages, 1676, whose sequence is complementary to the apical stem-loop of HIV-1 TAR RNA, was prepared to determine its effects on Tat protein-TAR interaction and Tat-mediated gene transactivation in cell culture. This oligomer and its all-phosphodiester analogue, 1707, were shown to: (1) bind to TAR at 37 degrees C with K(d)'s in the low nM concentration range; (2) inhibit Tat-TAR complex formation; and (3) inhibit expression of a chloramphenicol reporter gene under control of the HIV LTR in HeLa HL3T1 cells in culture.

Base Sequence↗
PubMed2003

siRNA function in RNAi: a chemical modification analysis.

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.

HeLa Cells↗