Talks

Despite the presence and biological role of D-amino acids, the fundamental issue of how proteins are made only with the L-chiral entities was largely ignored. Over the last two decades, it has become clearer as to how multiple ‘Chiral Checkpoints’ work in concert to avoid D-amino acids from getting incorporated into proteins (1, 2). Our recent work has shed light on how chiral proofreading systems have played critical roles in important evolutionary transitions (3, 4). In the first part of my talk, I will introduce the work that came out of the laboratories of two of the pioneers of nucleic acid research in the area of protein biosynthesis, Paul Berg and Donald Crothers, more than half a century back. Their work on the identification of D-aminoacyl-tRNA deacylase (DTD) and ‘Discriminator hypothesis’, respectively, were hugely ahead of their time and were partly against the general paradigm at that time. In both of the above works, the smallest and the only achiral amino acid turned out to be an outlier as DTD can act weakly on glycine charged tRNAs with a unique discriminator base of ‘Uracil’. This peculiar nature of glycine remained an enigma for nearly half a century. With a load of available information on the subject by the turn of the century, our work on ‘chiral proofreading’ mechanisms during protein biosynthesis serendipitously led us to revisit these findings. Our analysis has uncovered an unexpected connection between them that has implications for evolution of different eukaryotic life forms (5) and will be the focus of the second part of my talk.
Selected references:
1. Kuncha, S. K. et al., J. Biol. Chem. 2019 (Review article).
2. Kumar, P. et al. FEBS Letters 2022 (Review article).
3. Gogoi, J. et al. Sci. Adv. 2022.
4. Kumar, P. et al. Proc. Natl. Acad. Sci. (USA) 2023.
5. Kumar, P. and Sankaranarayanan, R. NAR 2024 (Critical Reviews and Perspective article).

Despite the presence and biological role of D-amino acids, the fundamental issue of how proteins are made only with the L-chiral entities was largely ignored. Over the last two decades, it has become clearer as to how multiple ‘Chiral Checkpoints’ work in concert to avoid D-amino acids from getting incorporated into proteins (1, 2). Our recent work has shed light on how chiral proofreading systems have played critical roles in important evolutionary transitions (3, 4). In the first part of my talk, I will introduce the work that came out of the laboratories of two of the pioneers of nucleic acid research in the area of protein biosynthesis, Paul Berg and Donald Crothers, more than half a century back. Their work on the identification of D-aminoacyl-tRNA deacylase (DTD) and ‘Discriminator hypothesis’, respectively, were hugely ahead of their time and were partly against the general paradigm at that time. In both of the above works, the smallest and the only achiral amino acid turned out to be an outlier as DTD can act weakly on glycine charged tRNAs with a unique discriminator base of ‘Uracil’. This peculiar nature of glycine remained an enigma for nearly half a century. With a load of available information on the subject by the turn of the century, our work on ‘chiral proofreading’ mechanisms during protein biosynthesis serendipitously led us to revisit these findings. Our analysis has uncovered an unexpected connection between them that has implications for evolution of different eukaryotic life forms (5) and will be the focus of the second part of my talk.
Selected references:
1.    Kuncha, S. K. et al., J. Biol. Chem. 2019 (Review article).
2.    Kumar, P. et al. FEBS Letters 2022 (Review article).
3.    Gogoi, J. et al. Sci. Adv. 2022.
4.    Kumar, P. et al. Proc. Natl. Acad. Sci. (USA) 2023.
5.    Kumar, P. and Sankaranarayanan, R. NAR 2024 (Critical Reviews and Perspective article).
 

In RNA, the G:C base-pair is much stronger than the A:U base-pair. This results in a strong bias for the incorporation of G and C residues in nonenzymatic RNA template copying chemistry. However, work from the Sutherland group on potentially prebiotic nucleotide synthesis has suggested that the 2-thio pyrimidines were the (prebiotic) precursors of the canonical pyrimidines. We have found that the 2-thio-U:A and 2-thio-C:I base-pairs are isomorphic and isoenergetic. As a result, template copying with an alphabet of 2-thio-U, 2-thio-C, adenosine and inosine shows less bias in nucleotide incorporation, while maintaining good fidelity. The convergence of plausible synthetic pathways with optimal template copying chemistry suggests that modern RNA may have been preceded by a primordial version based on related but distinct nucleotides.

Beyond the transfer of genetic information, RNA drives many cellular processes, e.g., transcription, splicing, translation, and its own stability, through its three-dimensional structures. The RNA molecules adopt simple secondary to complex tertiary structures using Watson-Crick base-pairing and tertiary interactions such as loops, bulges, helical junctions, and long-range interactions. The RNA secondary and tertiary structures are in equilibrium with competitive alternative conformations to form a different population of substructures. The different RNA substructures give rise to a distinct biological outcome and play a crucial role in human health and disease. The alternative RNA conformations equilibrium can be shifted in response to external cues such as small molecule ligands for therapeutic and biotechnological applications. Our group research focuses on modulating the nucleic acids structure- mediated gene regulation by small molecules in humans, bacteria and viruses for biomedical applications. In the talk, I will provide an overview of some of the alternate RNA conformations, e.g., G-quadruplex, riboswitches, etc., and the use of these structures for therapeutic and biotechnological applications.

References:
1. Ojha D, Rode AB*, Nature Communications (2025) 16(1):5751.
2. Gupta P., Khadake R.M., Singh O.N., Mirgane, H.A., Gupta, D., Bhosale S.V., Vrati, S., Surjit, M., Rode A. B.* ACS Infect. Dis. (2025), 11, 3,
784–795.
3. Khadake R.M., Arora V., Gupta P., Rode A. B.* ChemBioChem (2025), e202401015.
4. Gupta P, Khadake RM, Panja S, Shinde K, Rode AB* Genes, (2022), 13, 1930.
5. Pandey M, Ojha D, Bansal S, Rode AB*, Chawla G* Molecular Aspects of Medicine (2021), 81, 101003.
 

A hallmark pathological feature of ALS and FTD is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. We have been systematically identifying cryptic splicing targets regulated by TDP-43 in human brain. We discovered that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we found that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function. We are currently exploring the function of UNC13A in ALS/FTD and characterizing several other novel cryptic splicing targets. Some of these represent powerful biomarkers and other ones might be therapeutic targets. We are also using genome wide approaches to identify genes that work with TDP-43 to regulate cryptic splicing. Many cryptic splicing events caused by TDP-43 loss lead to cryptic transcripts degraded by the RNA surveillance mechanism nonsense-mediated decay (NMD). Standard RNA-sequencing approaches miss many of these and we recently found a way to unmask them (by inhibiting NMD). In addition to cryptic splicing, we have also discovered loss of TDP-43 in FTD/ALS leads to widespread alternative polyadenylation changes, impacting expression of disease-relevant genes and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.

A hallmark pathological feature of ALS and FTD is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. We have been systematically identifying cryptic splicing targets regulated by TDP-43 in human brain. We discovered that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we found that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function. We are currently exploring the function of UNC13A in ALS/FTD and characterizing several other novel cryptic splicing targets. Some of these represent powerful biomarkers and other ones might be therapeutic targets. We are also using genome wide approaches to identify genes that work with TDP-43 to regulate cryptic splicing. Many cryptic splicing events caused by TDP-43 loss lead to cryptic transcripts degraded by the RNA surveillance mechanism nonsense-mediated decay (NMD). Standard RNA-sequencing approaches miss many of these and we recently found a way to unmask them (by inhibiting NMD).  In addition to cryptic splicing, we have also discovered loss of TDP-43 in FTD/ALS leads to widespread alternative polyadenylation changes, impacting expression of disease-relevant genes and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.