Sivakumar Vadivel Gnanasundram

Umeå University

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Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Journal of General Virology (2009)

Nithya Subramanian Prashant Mani Swagata Roy Sivakumar Vadivel Gnanasundram Debi P. Sarkar

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Replicon

Sendai virus

NS2-3 protease

10.1099/vir.0.010579-0

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Citations (22)

PI3Kδ activates E2F1 synthesis in response to mRNA translation stress

Nature Communications (2017)

Sivakumar Vadivel Gnanasundram Slovénie Pyndiah Chrysoula Daskalogianni Kate Armfield Karin Nylander

The c-myc oncogene stimulates ribosomal biogenesis and protein synthesis to promote cellular growth. However, the pathway by which cells sense and restore dysfunctional mRNA translation and how this is linked to cell proliferation and growth is not known. We here show that mRNA translation stress in cis triggered by the gly-ala repeat sequence of Epstein-Barr virus (EBV)-encoded EBNA1, results in PI3Kδ-dependent induction of E2F1 mRNA translation with the consequent activation of c-Myc and cell proliferation. Treatment with a specific PI3Kδ inhibitor Idelalisib (CAL-101) suppresses E2F1 and c-Myc levels and causes cell death in EBNA1-induced B cell lymphomas. Suppression of PI3Kδ prevents E2F1 activation also in non-EBV-infected cells. These data illustrate an mRNA translation stress-response pathway for E2F1 activation that is exploited by EBV to promote cell growth and proliferation, offering new strategies to treat EBV-carrying cancers.

E2F1

ribosome biogenesis

10.1038/s41467-017-02282-w

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Citations (24)

The p53 endoplasmic reticulum stress-response pathway evolved in humans but not in mice via PERK-regulated p53 mRNA structures

Cell Death and Differentiation (2023)

Leila Fusée Norman Salomao Anand Ponnuswamy Lixiao Wang Ignacio López

Abstract Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.

Integrated stress response

Translational regulation

P-bodies

Cellular stress response

eIF2

10.1038/s41418-023-01127-y

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Citations (7)

Two molecules of Has1 RNA helicase function simultaneously in the biogenesis of small and large ribosomal subunits

bioRxiv (Cold Spring Harbor Laboratory) (2019)

Sivakumar Vadivel Gnanasundram Isabelle C. Kos‐Braun Martin Koš

ABSTRACT The RNA helicase Has1 is involved in the biogenesis of both small and large ribosomal subunits. How it performs these separate roles is not fully understood. Here we provide evidence that two molecules of Has1 are recruited and temporarily present in the same time in 90S pre-ribosomes. We identified multiple Has1 binding sites in the 18S, 5.8S and 25S rRNAs. We show that while the Has1 catalytic activity is not required for binding to 5.8S/25S region in pre-rRNA, it is essential for binding to 18S sites. After the cleavage of pre-rRNA at the site A2 Has1 remains associated not only with pre-60S but unexpectedly also with the pre-40S ribosomes. The recruitment to 90S/pre-40S and pre-60S ribosomes is mutually independent. Our data reconcile some seemingly contradictory observations about Has1 function in ribosome biogenesis.

ribosome biogenesis

Eukaryotic Ribosome

RNA Helicase A

10.1101/594762

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Citations (0)

At least two molecules of the RNA helicase Has1 are simultaneously present in pre-ribosomes during ribosome biogenesis

Nucleic Acids Research (2019)

Sivakumar Vadivel Gnanasundram Isabelle C. Kos‐Braun Martin Koš

The RNA helicase Has1 is involved in the biogenesis of both small and large ribosomal subunits. How it performs these separate roles is not fully understood. Here we provide evidence that at least two molecules of Has1 are temporarily present at the same time in 90S pre-ribosomes. We identified multiple Has1 binding sites in the 18S, 5.8S and 25S rRNAs. We show that while the Has1 catalytic activity is not required for binding to 5.8S/25S region in pre-rRNA, it is essential for binding to 18S sites. After the cleavage of pre-rRNA at the A2 site, Has1 remains associated not only with pre-60S but, unexpectedly, also with pre-40S ribosomes. The recruitment to 90S/pre-40S and pre-60S ribosomes is mutually independent. Our data provides insight into how Has1 performs its separate functions in the synthesis of both ribosomal subunits.

Eukaryotic Ribosome

ribosome biogenesis

RNA Helicase A

10.1093/nar/gkz767

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Citations (14)

DNA and RNA Binding Proteins: From Motifs to Roles in Cancer

International Journal of Molecular Sciences (2022)

Ondřej Bonczek Wang Lixiao Sivakumar Vadivel Gnanasundram Sa Chen Lucia Hároníková

DNA and RNA binding proteins (DRBPs) are a broad class of molecules that regulate numerous cellular processes across all living organisms, creating intricate dynamic multilevel networks to control nucleotide metabolism and gene expression. These interactions are highly regulated, and dysregulation contributes to the development of a variety of diseases, including cancer. An increasing number of proteins with DNA and/or RNA binding activities have been identified in recent years, and it is important to understand how their activities are related to the molecular mechanisms of cancer. In addition, many of these proteins have overlapping functions, and it is therefore essential to analyze not only the loss of function of individual factors, but also to group abnormalities into specific types of activities in regard to particular cancer types. In this review, we summarize the classes of DNA-binding, RNA-binding, and DRBPs, drawing particular attention to the similarities and differences between these protein classes. We also perform a cross-search analysis of relevant protein databases, together with our own pipeline, to identify DRBPs involved in cancer. We discuss the most common DRBPs and how they are related to specific cancers, reviewing their biochemical, molecular biological, and cellular properties to highlight their functions and potential as targets for treatment.

10.3390/ijms23169329

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Citations (5)

A single synonymous mutation determines the phosphorylation and stability of the nascent protein

Journal of Molecular Cell Biology (2018)

Konstantinos Karakostis Sivakumar Vadivel Gnanasundram Ignacio López Aikaterini Thermou Lixiao Wang

Abstract p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

Polysome

Ribosomal protein

10.1093/jmcb/mjy049

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Citations (37)

MDM2’s dual mRNA binding domains co-ordinate its oncogenic and tumour suppressor activities

Nucleic Acids Research (2020)

Sivakumar Vadivel Gnanasundram Laurence Malbert-Colas Sa Chen Leila Fusée Chrysoula Daskalogianni

Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2's binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2's activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.

10.1093/nar/gkaa431

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Citations (13)

Cryptic AUG is important for 48S ribosomal assembly during internal initiation of translation of coxsackievirus B3 RNA

Journal of General Virology (2011)

Bhupendra Verma Anand Ponnuswamy Sivakumar Vadivel Gnanasundram Saumitra Das

We have investigated the possible role of a conserved cis-acting element, the cryptic AUG, present in the 5' UTR of coxsackievirus B3 (CVB3 ) RNA. CVB3 5' UTR contains multiple AUG codons upstream of the initiator AUG, which are not used for the initiation of translation. The 48S ribosomal assembly takes place upstream of the cryptic AUG. We show here that mutation in the cryptic AUG results in reduced efficiency of translation mediated by the CVB3 IRES; mutation also reduces the interaction of mutant IRES with a well characterized IRES trans-acting factor, the human La protein. Furthermore, partial silencing of the La gene showed a decrease in IRES activity in the case of both the wild-type and mutant. We have demonstrated here that the interaction of the 48S ribosomal complex with mutant RNA was weaker compared with wild-type RNA by ribosome assembly analysis. We have also investigated by chemical and enzymic modifications the possible alteration in secondary structure in the mutant RNA. Results suggest that the secondary structure of mutant RNA was only marginally altered. Additionally, we have demonstrated by generating compensatory and non-specific mutations the specific function of the cryptic AUG in internal initiation. Results suggest that the effect of the cryptic AUG is specific and translation could not be rescued. However, a possibility of tertiary interaction of the cryptic AUG with other cis-acting elements cannot be ruled out. Taken together, it appears that the integrity of the cryptic AUG is important for efficient translation initiation by the CVB3 IRES RNA.

Eukaryotic translation

Eukaryotic initiation factor

Initiation factor

Ribosomal binding site

10.1099/vir.0.032151-0

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Citations (12)

Targeting Ribosome assembly on the HCV RNA using a small RNA molecule

RNA Biology (2012)

Prasanna Bhat Sivakumar Vadivel Gnanasundram Prashant Mani Partho Sarothi Ray Debi P. Sarkar

Translation initiation of hepatitis C Virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the Internal Ribosome Entry Site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

5.8S ribosomal RNA

10.4161/rna.21208

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Citations (10)