Biswadip Das

Bio: Biswadip Das is an academic researcher from Jadavpur University. The author has contributed to research in topics: Messenger RNA & mRNA surveillance. The author has an hindex of 17, co-authored 39 publications receiving 1194 citations. Previous affiliations of Biswadip Das include University of Rochester & University of Florida.

Action of BTN1, the yeast orthologue of the gene mutated in Batten disease.

Abstract: Neuronal ceroid-lipofuscinoses (NCL) are autosomal recessive disorders that form the most common group of progressive neurodegenerative diseases in children, with an incidence as high as 1 in 12,500 live births, and with approximately 440,000 carriers in the United States1,2. Disease progression is characterized by a decline in mental abilities, increased severity of untreatable seizures, blindness, loss of motor skills and premature death. The CLN3 gene, which is responsible for Batten disease, has been positionally cloned3. The yeast gene, denoted BTN1, encodes a non-essential protein that is 39% identical and 59% similar to human CLN3 ( ref. 4). Strains lacking Btn1p, btn1-Δ, are resistant to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP) in a pH-dependent manner5. This phenotype was complemented by expression of human CLN3, demonstrating that yeast Btn1p and human CLN3 share the same function5. Here, we report that btn1-Δ yeast strains have an abnormally acidic vacuolar pH in the early phases of growth. Furthermore, DNA microarray analysis of BTN1 and btn1-Δ strains revealed differential expression of two genes, with at least one, HSP30, involved in pH control. Because Btn1p is located in the vacuole, we suggest that Batten disease is caused by a defect in vacuolar (lysosomal) pH control. Our findings draw parallels between fundamental biological processes in yeast and previously observed characteristics of neurodegeneration in humans.

N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases.

Abstract: N6-methyladenosine (m(6) A) modification in mRNA is extremely widespread, and functionally modulates the eukaryotic transcriptome to influence mRNA splicing, export, localization, translation, and stability. Methylated adenines are present in a large subset of mRNAs and long noncoding RNAs (lncRNAs). Methylation is reversible, and this is accomplished by the orchestrated action of highly conserved methyltransferase (m(6) A writer) and demethylase (m(6) A eraser) enzymes to shape the cellular 'epitranscriptome'. The engraved 'methyl code' is subsequently decoded and executed by a group of m(6) A reader/effector components, which, in turn, govern the fate of the modified transcripts, thereby dictating their potential for translation. Reversible mRNA methylation thus adds another layer of regulation at the post-transcriptional level in the gene expression programme of eukaryotes that finely sculpts a highly dynamic proteome in order to respond to diverse cues during cellular differentiation, immune tolerance, and neuronal signalling.

Degradation of normal mRNA in the nucleus of Saccharomyces cerevisiae.

Abstract: The rate of synthesis of a protein is determined primarily by the steady-state level of the corresponding mRNA, which, in turn, is determined by the rate of synthesis and degradation of the mRNA. Thus, mRNA stability is an important parameter in the regulation of gene expression, affecting both the steady-state level of the protein and the transient time of translation of the formed transcript. Normal mature mRNAs of Saccharomyces cerevisiae are degraded through a major 5′-to-3′ (5′→3′) pathway and a minor 3′→5′ pathway, both of which take place in the cytoplasm. Both degradation pathways begin with the shortening of the poly(A) tail to a track of A10 or less, caused by a Pop2p-Ccr4p-Caf1p poly(A) nuclease complex (16, 73). In the major pathway, deadenylation causes disassociation of the Pab1p [poly(A) binding protein] from the cap binding protein eIF4G, followed by the removal of the 5′ cap by the decapping enzyme Dcp1p. Subsequently, the decapped mRNA is rapidly degraded by the 5′→3′ exonuclease Xrn1p and by assistance of Sbp8p and other protein components (4, 5, 17, 32, 47, 57, 58, 71). In the minor pathway, deadenylated mRNAs are subjected to 3′→5′ degradation by the action of the exosome, a complex of 10 3′→5′ riboexonucleases that also plays a central role in the precise formation of the 3′ ends of several types of RNAs (9), including processing precursors for rRNAs in the nucleus. The exosome may also degrade fragments of mRNA released by endonucleolytic cleavage. Yeast mutants lacking either exonucleolytic pathway degrade their mRNAs more slowly, but the loss of both pathways is lethal (54). While most mRNAs are slowly deadenylated before rapid degradation, certain normal and mutant mRNAs are rapidly degraded by a third specialized pathway, known as nonsense-mediated mRNA decay (NMD) pathway, or mRNA surveillance, which triggers decapping before deadenylation (26, 29, 55). Substrates of the NMD pathway include not only mRNAs containing nonsense mutations but also wild-type mRNAs that contain the following: inefficiently spliced pre-mRNAs that enter the cytoplasm upstream open reading frames (14, 77) and certain codons subject to leaky scanning (80). In fact, Lelivelt and Culbertson (48) showed that mutation of protein components of NMD could actually lead to the increase in the steady-state levels of a wide spectrum of normal mRNAs. The NMD pathway discriminates between nonsense codons on the basis of downstream sequence elements located 3′ to susceptible nonsense codons (60, 87). In addition, yeast has the capacity to recognize and degrade mRNAs lacking all termination codons, a process that occurs by a mechanism distinct from NMD and from the major mRNA turnover pathway that requires deadenylation, decapping, and 5′→3′ exonucleolytic decay (20, 76). Previously, Das et al. (15) presented preliminary evidence, based on the analysis of cyc1-512 suppressors, that Cbc1p, the large subunit of nuclear cap binding complex, is involved in a novel mRNA degradation system. The cyc1-512 mutation causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3′ end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs, which differ in length at their 3′ termini (15, 85). Suppression analysis of cyc1-512 showed that it can be suppressed by deletion of either of the nonessential genes CBC1 or CBC2, which encode, respectively, the CBP80 or CBP20 subunits of the nuclear cap binding complex, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex responsible for NMD. Suppression of cyc1-512 by cbc1-Δ occurred by two different mechanisms. The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Δ mutants by promoting 3′-end formation at otherwise weak sites; whereas the levels of the longer cyc1-512 transcripts, as well as all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Δ greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain that is defective in mRNA export. These findings led Das et al. (15) to suggest that Cbc1p possibly defines a novel degradation pathway that acts on mRNAs partially retained in nuclei. However, the interpretation of the results obtained with rat7-1 was complicated by the suppression of the mRNA export defect by cbc1-Δ, allowing growth at the restrictive temperature, and thus preventing meaningful studies with mRNA half-lives and in situ mRNA localization using fluorescence in situ hybridization (FISH). Thus, it remained to be definitively established if the Cbc1p-dependent mRNA decay system was located in the nucleus. In this study, we definitely established the existence of this novel mRNA degradation pathway which we named the DRN (for decay of RNA in the nucleus) pathway, and we conclusively confirmed the involvement of Cbc1p in this pathway. We have investigated the nature of this degradation pathway, primarily by using a mutation (nup116-Δ) in NUP116 which encodes a nucleoporin that plays a central role in nuclear mRNA export (3, 31). nup116-Δ strains grow slowly at 25°C and are inviable at 37°C (81). The lethal phenotype correlates with defects in mRNA export and perturbations of structures of the nuclear envelope and nuclear pore complexes, resulting in the complete nuclear accumulation of mRNA (82). We show that retention of mRNAs in the nucleus causes accelerated degradation of representative transcripts. Deletions of either CBC1 or RRP6, which encodes a nuclear 3′→5′ exoribonuclease associated with the exosome, suppressed the rapid mRNA degradation phenotype. Deletion of RAI1, which encodes a nuclear protein required for the activity of the nuclear 5′→3′ exoribonuclease Rat1p, also suppressed the rapid degradation, but to a lesser extent. We conclude that DRN involves the Rrp6p and Rat1p nuclear exonucleases, as well as the CBC, the nuclear cap binding complex, which may direct the mRNAs to the site of degradation.

In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: the role of the 50S particle and its 23S rRNA

Abstract: Ribosomes from a number of prokaryotic and eukaryotic sources (e.g., Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HCl prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydro-genase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver, The protein-folding activity of E. coli, ribosomes was found to be present in 50s particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.

The Role of Nuclear Cap Binding Protein Cbc1p of Yeast in mRNA Termination and Degradation

Abstract: The cyc1-512 mutation in Saccharomyces cerevisiae causes a 90% reduction in the level of iso-1-cytochrome c because of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly long cyc1-512 mRNAs which differ in length at their 3' termini. cyc1-512 can be suppressed by deletion of either of the nonessential genes CBC1 and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-Delta deletion suppressed the cyc1-512 defect by diminishing degradation of the longer subset of cyc1-512 mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512 defects by cbc1-Delta occurred by two different mechanisms. The levels of the shorter cyc1-512 transcripts were enhanced in the cbc1-Delta mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longer cyc1-512 transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore, cbc1-Delta greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1 strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

Molecular chaperones in cellular protein folding.

Abstract: The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.

Integrative Genomics Viewer

Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

The highways and byways of mRNA decay.

Abstract: Turnover of mRNA is a key mechanism in regulated gene expression. In addition to turnover pathways for normal transcripts, there are surveillance mechanisms that degrade aberrant mRNAs. mRNA decay is regulated in response to cellular signals and coordinated with other mRNA-metabolic processes. When considering the control of gene expression, the focus has traditionally been on transcriptional regulation. Recently, however, the large contribution made by mRNA decay has become difficult to ignore. Large-scale analyses indicate that as many as half of all changes in the amounts of mRNA in some responses can be attributed to altered rates of decay. In this article, we discuss some of the mechanisms that are used by the cell to mediate and regulate this intriguing process.

The Nonsense-Mediated Decay RNA Surveillance Pathway

Abstract: Nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that selectively degrades mRNAs harboring premature termination (nonsense) codons. If translated, these mRNAs can produce truncated proteins with dominant-negative or deleterious gain-of-function activities. In this review, we describe the molecular mechanism of NMD. We first cover conserved factors known to be involved in NMD in all eukaryotes. We then describe a unique protein complex that is deposited on mammalian mRNAs during splicing, which defines a stop codon as premature. Interaction between this exon-junction complex (EJC) and NMD factors assembled at the upstream stop codon triggers a series of steps that ultimately lead to mRNA decay. We discuss whether these proofreading events preferentially occur during a “pioneer” round of translation in higher and lower eukaryotes, their cellular location, and whether they can use alternative EJC factors or act independent of the EJC.