59 research outputs found
Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA
No full textThe HIV regulatory proteins Tat and Rev have a nucleolar localization property in human cells. However, no functional role has been attributed to this localization. Recently it has been demonstrated that expression of Rev induces nucleolar relocalization of some protein factors involved in Rev export. Because the function of Rev is to bind HIV RNA and facilitate transport of singly spliced and unspliced RNA to the cytoplasm, it is likely that the nucleolus plays a critical role in HIV-1 RNA export. As a test for trafficking of HIV-1 RNAs into the nucleolus, a hammerhead ribozyme that specifically cleaves HIV-1 RNA was inserted into the body of the U16 small nucleolar RNA, resulting in accumulation of the ribozyme within the nucleoli of human cells. HeLa CD4(+) and T cells expressing this nucleolar localized ribozyme exhibit dramatically suppressed HIV-1 replication. The results presented here suggest a trafficking of HIV-1 RNA through the nucleoli of human cells, thus posing a different paradigm for lentiviral RNA processing
A nucleolar TAR decoy inhibitor of HIV-1 replication
No full textTat is a critical regulatory factor in HIV-1 gene expression. It mediates the transactivation of transcription from the HIV-1 LTR by binding to the transactivation response (TAR) element in a complex with cyclin T1. Because of its critical and early role in HIV gene expression, Tat and its interaction with the TAR element constitute important therapeutic targets for the treatment of HIV-1 infection. Based on the known nucleolar localization properties of Tat, we constructed a chimeric small nucleolar RNA-TAR decoy that localizes to the nucleoli of human cells and colocalizes in the nucleolus with a Tat-enhanced GFP fusion protein. When the chimeric RNA was stably expressed in human T lymphoblastoid CEM cells it potently inhibited HIV-1 replication. These results demonstrate that the nucleolar trafficking of Tat is critical for HIV-1 replication and suggests a role for the nucleolus in HIV-1 viral replication
U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus
No full textThe in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector
A critical role for the RNA m6A methylation complex in myeloid leukemia
No full textN6-methyladenosine (m6A) is a well-known RNA modification that can affect mRNA splicing, stability and translation (D. Dominissini et al., 2012; X. Wang et al. 2015; Y. Wang et al. 2014; J. Zhou et al. 2015; J. Choi et al. 2016), and the processing of miRNA precursors (C.R. Alarcon et al. 2015). In mammals, the m6A writer is a multicomponent complex composed of the two methyltransferases METTL3 and METTL14 (J. Liu et al. 2014), and the regulatory protein WTAP (XL. Ping et al. 2014). Post-transcriptional m6A RNA modification is indispensable for cell viability and development, yet its role in cell differentiation and disease are still poorly understood. Notably, WTAP protein is an oncogene overexpressed in Acute Myeloid Leukemia (AML) compared to healthy control cells (H. Bansal et al., 2014). Moreover, METTL3, METTL14, and RBM15 are highly expressed in AML compared with other cancers (S.R. Jaffrey and M G. Kharas, 2017). We analysed the expression of the m6A RNA methylation complex components in AML observing that both METTL3 and METTL14 mRNAs are upregulated in AML samples (TCGA) respect to fully differentiated myeloid cells (GeoDatasets). Conversely, WTAP mRNA has lower levels in AML, even if the protein is upregulated. We showed that in AML and Chronic Myeloid Leukemia (CML) cell lines METTL3 is localized in both nucleus and cytoplasm. In cytoplasm METTL3 binds WTAP mRNA enhancing its translation independently from its catalytic activity, possibly explaining, at least partially, WTAP protein increased levels observed in AML cells. Moreover, we investigated the role of m6A RNA methylation complex components in myeloid leukemia cells, confirming that WTAP affects proliferation and differentiation of AML cells acting as an oncogene and demonstrating that METTL3 and METTL14 are essential proteins in these cells. Indeed, depletion of the two methyltransferases leads to a marked cellular mortality of AML cells. Notably, METTL3 affects viability and proper myeloid differentiation also in hematopoietic stem cells CD34+ derived from healthy umbilical cord. In CML K562 cells METTL3 and METTL14 depletion does not lead to apoptosis but could affect proper ribosome biogenesis, leading to an evident slowdown of cellular proliferation. Notably, decreased levels of METTL3 and METTL14 in K562 cells cause a downregulation of MYC protein, as recently reported in AML cells (L.P. Vu et al., 2017). Analysing ENCODE MYC ChIP- seq data we observed strong MYC peaks on the promoter of all the genes critical for ribosome biogenesis that are affected by METTL3 and METTL14 depletion, suggesting a MYC-mediated effect. In conclusion, we analysed some functional aspects of m6A RNA chemical modification in both acute and chronic myeloid leukemia cells, identifying differential m6A-regulated pathways between these two kinds of pathologies. Moreover, we also highlighted a more complex and double role for METTL3 protein in these cells, being one of the two essential m6A methyltransferase in the nucleus, and an RNA binding protein promoting translation in cytoplasm. Altogether, these data indicate a link between m6A factors and leukemogenesis and pave the way to possible future therapies targeting the RNA m6a methylation complex components
Two different snoRNAs are encoded in introns of amphibian and human L1 ribosomal protein genes
No full textWe previously reported that the third intron of the X.laevis L1 ribosomal protein gene encodes for a snoRNA called U16. Here we show that four different introns of the same gene contain another previously uncharacterized snoRNA (U18) which is associated with fibrillarin in the nucleolus and which originates by processing of the pre-mRNA. The pathway of U18 RNA release from the pre-mRNA is the same as the one described for U16: primary endonucleolytic cleavages upstream and downstream of the U18 coding region produce a pre-U18 RNA which is subsequently trimmed to the mature form. Both the gene organization and processing of U18 are conserved in the corresponding genes of X.tropicalis and H.sapiens. The L1 gene thus has a composite structure, highly conserved in evolution, in which sequences coding for a ribosomal protein are intermingled with sequences coding for two different snoRNAs. The nucleolar localization of these different components suggests some common function on ribosome biosynthesis
Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection
No full textAdenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4(+) T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5' untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1
Use of adenoviral VAI small RNA as a carrier for cytoplasmic delivery of ribozymes
No full textThe in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several features: RNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of "natural" small RNA coding genes as expressing cassettes gives high chances to fulfill these requirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1. The conserved 5' leader sequence of HIV was chosen as a target, because it is present in all the viral transcripts and is highly conserved. Hammerhead ribozymes were substituted to different portions of the VAI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compartmentalization, and assembly in specific ribonucleoparticles containing the La antigen. Interesting differences in the activity of the different chimera were found in both in vitro cleavage assays and S100 extracts of injected oocytes where the catalytic activity of the ribozymes in the RNP context can be analyzed
The Rev protein is able to transport to the cytoplasm small nucleolar RNAs containing a Rev binding element
No full textSmall nucleolar RNAs (snoRNAs) were utilized to express Rev-binding sequences inside the nucleolus and to test whether they are substrates for Rev binding and transport. We show that U16 snoRNA containing the minimal binding site for Rev stably accumulates inside the nucleolus maintaining the interaction with the basic C/D snoRNA-specific factors. Upon Rev expression, the chimeric RNA is exported to the cytoplasm, where it remains bound to Rev in a particle devoid of snoRNP-specific factors. These data indicate that Rev can elicit the functions of RNA binding and transport inside the nucleolus
A novel small nucleolar RNA (U16) is encoded inside a ribosomal protein intron and originates by processing of the pre-mRNA
No full textWe report that the third intron of the L1 ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre-mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human L1 ribosomal protein gene a highly homologous region is found which can be released from the pre-mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the L1 ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components
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