Haoquan Wu

• Texas Tech University Health Sciences Center

5′-isomiRs expand the repertoire of miRNA targets. However, how they are generated is not well understood. Previously, we showed that for some miRNAs in mammalian cells, Drosha cleaves at multiple sites to generate multiple pre-miRNAs that give rise to multiple 5′-isomiRs. Here, we showed that for some other miRNAs, 5′-isomiRs are generated by alte...

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors...

RNA interference (RNAi) provides a powerful tool to silence specific gene expression and has been widely used to suppress host factors such as CCR5 and/or viral genes involved in HIV-1 replication. Newer nuclease-based gene-editing technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the clu...

Background: Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a con...

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fid...

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes i...

Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects...

Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficien...

Relatively large amounts of transfected siRNA can compete for Ago proteins and thus compromise endogenous miRNA function, potentially leading to toxicities. Here, we show that shRNA can also perturb endogenous miRNA function similarly. More importantly, we also show that the problem can be solved by designing shRNAs in the context of pre-miR-451 st...

Pol III promoters such as U6 are commonly used to express small RNAs, including small interfering RNA, short hairpin RNA, and guide RNA, for the clustered regularly interspaced short palindromic repeats genome-editing system. However, whether the small RNAs were precisely expressed as desired has not been studied. Here, using deep sequencing to ana...

Significance MicroRNA genes are transcribed as long primary microRNAs (pri-miRNAs) and then processed by the Drosha–DGCR8 (DiGeorge syndrome critical region gene 8) complex into ∼60-nt pre-miRNAs, which is the key step of miRNA biogenesis. How exactly the Drosha–DGCR8 complex recognizes pri-miRNAs and selects the processing site is not well underst...

Hypersecretion of cytokines by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here, we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely imm...

Mismatches increase functionality. Three siRNAs that did not effectively inhibit West Nile virus replication in our previous study (Figure 1a of reference (31)) were redesigned by increasing the length to 22 nt and introducing mismatches in the passenger strand corresponding to guide strand position 1 and 12 (m1+12) and tested for functionality as...

siRNA targeting highly conserved regions in the HIV 5′UTR with and without mismatches were tested for efficacy as in Fig. 1 . (TIF)

The sequences of various mismatched siRNAs. “AS” represent the guide strand while “S” represent the passenger strand. (XLS)

DNA oligos used to construct psiCHECH2 vector to determine siRNA repression efficiency. The forward and reverse strands were annealed and cloned into psiCHECK2. (XLS)

shRNA sequences. Mature duplex sequences are marked in bold. Small cap represents the mismatched nucleotides. (XLS)

siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and t...

RNA interference can be mediated by fully complementary siRNA or partially complementary miRNA. siRNAs are widely used to suppress viral replication and the fully complementary siRNA bound Ago-2 in the RISC is known to degrade the target RNA. Although other argonaute proteins lacking slicer activity can also bind oligonucleotides with both si and m...

West Nile (WN) and St. Louis encephalitis (SLE) viruses can cause fatal neurological infection and currently there is neither a specific treatment nor an approved vaccine for these infections. In our earlier studies, we have reported that siRNAs can be developed as broad-spectrum antivirals for the treatment of infection caused by related viruses a...

MicroRNAs have emerged as - important posttranscriptional regulators of gene expression. Small RNA cloning is a powerful method to identify new microRNAs (miRNAs) and to profile miRNA expression. In addition, it reveals end heterogeneity that may be important in miRNA function. Here, we describe a protocol that is optimized to clone small RNAs from...

Inflammation mediated by tumor necrosis factor-alpha (TNF-alpha) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-alpha in the central nervous system (CNS). Here, we show that suppression of TNF-alpha by targeted delivery of small interf...

RNA interference (RNAi)-mediated knockdown of gene expression offers a novel treatment strategy for human immunodeficiency virus (HIV) infection. However, the major hurdle for clinical use is a practical strategy for small interfering RNA (siRNA) delivery to the multiple immune cell types important in viral pathogenesis. We have developed a novel i...

Inflammation mediated by TNF-a and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed the major source of TNF-a in the central nervous system. Here, we show that suppression of TNF-a by targeted delivery of siRNA to macrophage/microglial cells dramatically reduces LPS-indu...

Primers used for cloning the pre-miRNA ends. (0.05 MB DOC)

It is generally believed that the miRNA processing machinery ensures the generation of a mature miRNA with a fixed sequence, particularly at its 5' end. However, we and others have recently noted that the ends of a given mature miRNA are not absolutely fixed, but subject to variation. Neither the significance nor the mechanism behind the generation...

In less than a decade after discovery, RNA interference-mediated gene silencing is already being tested as potential therapy in clinical trials for a number of diseases. Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being in...

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD...

Real time RT-PCR to detect lower frequency miRNAs. Expression of indicated miRNAs in T cell subsets was tested by real time PCR. Expression level was normalized to that of small non-coding RNA U6B. Mean of triplicate experiments±SD is shown. (0.21 MB TIF)

Sequence of all miRNAs cloned in naïve, effector and memory T cells. Annotated miRNA sequences extracted from the concatemerized small RNA sequencing in different T cell subsets is shown. Only miRNAs cloned more than 4 times are included. The numbers before each sequence represent individual identity marks. (0.08 MB XLS)

Primers used for the 3′RACE-based real time PCR. (0.01 MB XLS)

miRNAs cloned from naïve, effector and memory T cells. The cloning frequency of individual microRNAs and their representation as a percentage of total miRNA clones isolated in each T cell subset are shown. (0.02 MB XLS)

Microarray analysis of cloned miRNAs also reveals differential expression in T cell subsets. Hybridization signals obtained in the microarray for miRNAs that were identified by direct cloning are shown. (0.02 MB XLS)

Normalized miRNA cloning frequency in T cell subsets. The percent cloning frequency in Supplemental Table 1 was normalized according to total hybridization signal obtained in the microarray for the corresponding T cell subset to obtain the normalized expression data. Only miRNAs cloned more than 4 times are shown. (0.02 MB XLS)

A major impediment in the treatment of neurological diseases is the presence of the blood-brain barrier, which precludes the entry of therapeutic molecules from blood to brain. Here we show that a short peptide derived from rabies virus glycoprotein (RVG) enables the transvascular delivery of small interfering RNA (siRNA) to the brain. This 29-amin...

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific naïve, effector...

bri3 was identified to be a novel gene up-regulated in TNF-treated cells with suppressed subtractive hybridization (SSH) in our laboratory. Previous studies showed that overexpression of BRI3 induced apoptosis in L929 cells. To further study the function of bri3, we disrupted its expression by expressing bri3 antisense RNA. The antisense RNA promot...

Neural stem cells are the multipotential, self-renewing cells in central nerve system, and play an essential role in the development and differentiation of nerve system. Neural stem cells can be used to treat the nerve system diseases, especially, the transplantation of neural stem cells to rescue the degenerated neural cells has become a very prom...