Welcome to the laboratory of Richard J. Maraia, M.D.
Section on Molecular and Cell Biology, Intramural Research Program in Genomics of Differentiation,
Eunice Kennedy Shriver National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, MD, USA
We are a basic research laboratory of Molecular Biology and Genetics that seeks to understand the influences of genetics, biochemistry and cell biology on the metabolism of small nuclear RNAs, tRNAs and messenger RNAs, and how these contribute to growth and development. Of interest is the human La antigen and La-related protein-4 (LARP4) in their ability to accommodate specific binding to precursor-tRNAs and mRNAs and influence their 3' end metabolism in the nucleus and cytoplasm respectively. A major interest is in the biogenesis and metabolism of the transfer RNAs, the genetic adapters that translate the genetic code, and the influences of their dynamics on codon bias-driven genetic programs involved in growth and in response to physiologic stress and disease. An active project is to understand the role of the anticodon loop modification that occurs on a small subset of tRNAs, the enzymes that mediate it, their roles in physiology and the consequences of their dysfunction. One such modification is that mediated by the conserved family of tRNA isopentenylytransferases (IPTases), which in humans is known as TRIT1. These enzymes add an isopentyl group to the N6 atom of adenosine-37 of a subset of cytosolic and mitochondrial tRNAs that decode codons that begin with U. Hereditary mutations that impair the tRNA modification activity of TRIT1 lead to neurodevelopmental disorders in children.
Our group also investigates the molecular mechanisms involved in RNA synthesis by RNA polymerase III, which serves as a model system of eukaryotic transcription, as well as the post-transcriptional phase of RNA processing, transcript maturation, degradation and decay. Pol III subunits C37, C53 are actively under investigation, as is the RNA 3' cleavage factor subunit, C11. The C37/53 subunit heterodimer functions in transcription termination by controlling dynamics of the 3' end of the DNA:RNA in the polymerase active center.
Transcription termination by pol III is linked to posttranscriptional RNA processing because the mechanism of termination leads to synthesis of 3' oligo-U tracts on nascent transcripts, which serve as a sequence-specific binding site for the La protein. Thus, another focus is on the process by which a nascent tRNA precursor is converted to a mature functional tRNA. Our goal is to understand the mechanisms involved in these processes, their regulation, and how they impact upon cell growth in normal and pathological states of cellular proliferation. Other interests include mRNA metabolism involving LARP4 and its relationship to poly(A) binding protein (PABP) and mRNA poly(A) tail (PAT) homeostasis. We have shown that LARP4 expression of leads to mRNA PAT lengthening whereas genetic deletion of LARP4 leads to PAT shortening with decreased stability of the mRNAs.
Richard J Maraia, MD
B.S., Columbia University
M.D., Cornell University Medical College
Some methodologies that we developed and/or make regular use of:
•tRNA-mediated suppression (TMS); An in vivo system in the fission yeast, S. pombe that uses a suppressor-tRNASerUCA that can insert serine at a premature UGA stop codon in the ade6-704 mRNA and thereby prevent red pigment accumulation, providing a colorimetric colony assay. This has been used to examine numerous aspects of tRNA biogenesis including transcription by RNA polymerase III, posttranscriptional processing, nuclear export, tRNA modification and function at the ribosome.
(Described in a methods review article; Rijal et al. Gene. 2015 556:35–50. PMCID: PMC4272643Article PubReader PDF–1.2M, and recently further adapted/expanded by addition of a nanoluciferase-suppressible reporter of TMS for quantitiion (Sakhawala and Maraia, manuscript in preparation).
•PHA6 and PHA26 assays; These are tRNA base modification-specific and -sensitive northern blot methods that are used to assess the modification status of specific tRNA modifications on specific cellular tRNA species in a semiquantitative manner.
(Described in Lamichhane et al., RNA. 2011 17:1846-57. PMID:21873461 Free PMC Article, Lamichhane et al., Mol Cell Biol. 2013 33:2918-29. PMID:23716598 Free PMC Article, Lamichhane et al., Mol Cell Biol. 2013 33:4900-8. PMID:24126054 Free PMC Article, Arimbasseri et al., PLoS Genet. 2015 11(12):e1005671. PMID:26720005 Free PMC Article, Rojas-Benitez et al., J Biol Chem. 2015 290(30):18699-707. PMID:26063805 Free PMC Article, Lamichhane RNA. 2016 22:583-96. PMID:26857223 Free PMC Article, Rojas-Benitez et al., J Biol Chem. 2015 290(30):18699-707. PMID:26063805 Free PMC Article)
•tRNA-hydroSeq; Illumina based tRNA-sequencing that can be used to compare relative amounts of tRNAs from different samples and also captures tRNA modification information on five base modifications.
(Described in Arimbasseri et al., PLoS Genet. 2015 11(12):e1005671. PMID:26720005 Free PMC Article, Arimbasseri et al., RNA. 2016 22:1400-10. PMID:27354703 Free PMC Article, Mattijssen et al., Elife. 2017 12;6. pii: e28889. PMID:28895529 Free PMC Article)
•mPAT-Seq (mRNA polyA tail-Seq); Long read high throughput sequencing using PacBio Sequel system to read poly(A) tail length on mRNAs (Mattijssen et al., manuscript in preparation).