Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates

Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5′ and 3′ mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5′ mRNA fragment was more abundant and displayed a greater stability than the corresponding 3′ mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5′ mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5′ cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing

Prognostic Impact of Pattern of Mandibular Involvement in Gingivo-Buccal Complex Squamous Cell Carcinomas:Marrow and Mandibular Canal Staging System

PURPOSE: To study the pattern of mandibular involvement and its impact on oncologic outcomes in patients with gingivo-buccal complex squamous cell carcinoma (GBC-SCC) and propose a staging system based on the pattern of bone involvement (MMC: Marrow and mandibular canal staging system) and compare its performance with the 8th edition of the American Joint Committee on Cancer (AJCC8). METHODS: This retrospective observational study included treatment-naïve GBC-SCC patients who underwent preoperative computed tomography (CT) imaging between January 1, 2012, and March 31, 2016, at a tertiary care cancer center. Patients with T4b disease with high infratemporal fossa involvement, maxillary erosion, and follow-up of less than a year were excluded. The chi-square or Fisher’s exact test was used for descriptive analysis. Kaplan–Meier estimate and log-rank test were performed for survival analysis. Multivariate analysis was done using Cox regression analysis after making adjustments for other prognostic factors. p-Value <0.05 was considered as significant. Based upon the survival analysis with different patterns of bone invasion, a new staging system was proposed “MMC: Marrow and mandibular canal staging system”. “Akaike information criterion” (AIC) was used to study the relative fitted model of the various staging (TNM staging—AJCC8) with respect to survival parameters. RESULTS: A total of 1,200 patients were screened; 303 patients were included in the study. On radiology review, mandibular bone was involved in 62% of patients. The pattern of bone involvement was as follows: deep cortical bone erosion (DCBE) in 23%, marrow in 34%, and marrow with the mandibular canal in 43% of patients. Patients with DCBE and no bone involvement (including superficial cortical) had similar survival [disease-free survival (DFS) and locoregional recurrence-free survival (LRRFS)], and this was significantly better than those with marrow with or without mandibular canal involvement (for both DFS and LRRFS). Patients with DCBE were staged using the MMC, and when compared with the AJCC8, the MMC system was better for the prediction of survival outcomes, as AIC values were lower compared with those of the AJCC8. There was a significant association (p = 0.013) between the type of bone involvement and the pattern of recurrence. CONCLUSIONS: For GBC-SCC, only marrow with or without mandibular canal involvement is associated with poorer survival outcomes. As compared with the AJCC8, the proposed Mahajan et al. MMC staging system downstages DCBE correlates better with survival outcomes

2′-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2′-O-Methyl (2′-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2′-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types

RNA interference for the treatment of papillomavirus disease

Human Papillomavirus (HPV)-induced diseases are a significant burden on our healthcare system and current therapies are not curative. Vaccination provides significant prophylactic protection but effective therapeutic treatments will still be required. RNA interference (RNAi) has great promise in providing highly specific therapies for all HPV diseases yet this promise has not been realised. Here we review the research into RNAi therapy for HPV in vitro and in vivo and examine the various targets and outcomes. We discuss the idea of using RNAi with current treatments and address delivery of RNAi, the major issue holding back clinical adoption. Finally, we present our view of a potential path to the clinic

Predicting polymorphic EST-SSRs in silico

The public availability of large quantities of gene sequence data provides a valuable resource of the mining of Simple Sequence Repeat (SSR) molecular genetic markers for genetic analysis. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the characterization of barley EST-SSRs and the identification of putative polymorphic SSRs from EST data. Polymorphic SSRs are distinguished from monomorphic SSRs by the representation of varying motif lengths within an alignment of sequence reads. Two measures of confidence are calculated, redundancy of a polymorphism and co-segregation with accessions. The utility of this method is demonstrated through the discovery of 597 candidate polymorphic SSRs, from a total of 452 642 consensus expressed sequences. PCR amplification primers were designed for the identified SSRs. Ten primer pairs were validated for polymorphism in barley and for transferability across species. Analysis of the polymorphisms in relation to SSR motif, length, position and annotation is discussed

CD62L as a therapeutic target in chronic lymphocytic leukemia

Purpose: Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of CLL

Nuclear poly(A) tail size is regulated by Cnot1 during the serum response

The poly(A) tail removal from mRNAs introduces a delay between mRNA synthesis and decay. We measured levels and poly(A) tail sizes of serum-induced mRNAs and used mathematical modelling to compare their deadenylation time with the delay in decay and found that they are indeed correlated. Discrepancies between our data and the polyadenylation models at later time points after the peak of induction led us to investigate the size of the poly(A) tails on newly made mRNA. Surprisingly, new serum-induced mRNAs synthesised late in induction had short poly(A) tails (around A25) in the nucleus. In addition, newly made constitutive mRNAs had medium sized poly(A) tails (around A50). To see if deadenylation was responsible for the new short poly(A) tails, we depleted Cnot1, a subunit of the CCR4/NOT deadenylase. Cnot1 depletion led to slower deadenylation of cytoplasmic mRNAs, as expected, but also decreased transcription and led to longer nuclear mRNA poly(A) tails. These observations implicate CCR4/NOT in regulating both the transcription and the nuclear poly(A) tail size of serum-induced mRNAs. Detection of some chromatin-associated mRNAs with long poly(A) tails suggested that nuclear deadenylation is an early event. Our data show that initial poly(A) tail size of mRNAs can be regulated and is not always 200-250 nucleotides, adding a novel layer to the control of gene expression