Document text
Principal Investigator: Benedict Smail
Organization: DANA-FARBER CANCER INST
Fiscal Year: 2023
Award: $22,942
Funding agency: National Institute of General Medical Sciences
Project Summary
The objective of this research proposal is to characterize the enzymatic activities of the reverse transcriptase
(RT) of the human long interspersed element 1 (LINE-1, L1 RT). LINE-1 sequences constitute 17% of the
human genome, and L1 RT activity is responsible for approximately 40% of our genome because it has also
led to the proliferation of short interspersed elements (e.g., Alu), and other retroelements. L1 RT activity is also
implicated as a driving force behind a variety of human diseases such as macular degeneration, Aicardi-
Goutières syndrome, and systemic lupus erythematosus. LINE-1 expression and retrotransposition are
commonplace in numerous cancers, including 90-100% of breast, colon, and esophageal cancers, making
understanding the basic biochemical activity of L1 RT relevant to public health research.
Reverse transcription is key to the ability of LINE-1 to self-propagate in our genome, through a process
known as target-primed reverse transcription (TPRT). L1 RT is encoded by the second open reading frame
(ORF2) of L1, residing in the protein known as ORF2p. In this proposal, I show that I have isolated the ORF2p
RT domain, and that this has RT activity in vitro. I intend to use this protein domain, as well as full-length
ORF2p, and L1 RNPs to fully characterize the biochemical and enzymatic properties of L1 RT. In my first
Specific Aim, I will characterize its processivity, which is an indication of whether the RT stops and pauses, or
whether it keeps going until it reaches the end of a template. I will also measure the fidelity of L1 RT, which
defines how faithfully it copies DNA from an RNA template. Finally, I present preliminary sequencing data
showing that L1 RT has the unexpected ability to begin processing an RNA template without a primer and
propose rigorous experiments to study this feature. In my second Specific Aim, I focus on what happens when
L1 RT reaches the end of its template, which may be crucial to understanding how cells repair intermediates of
transposition. I present preliminary data showing that L1 RT adds extra nucleotides to the end of its cDNA and
propose studies to fully characterize this activity. Additionally, I will study the ability of L1 RT to switch to a
different template at the end of one template and whether LINE-1 RT can use modified RNA as a template,
such as the pseudouridine found in mRNA vaccines or the naturally occurring N6-methyladenosine
modification.
This work will advance our understanding of how LINE-1 operates inside cells and how LINE-1
completes its life cycle. The proposed research will have implications for cancer biology and innate immune
signaling where L1 RT is increasingly recognized as a DNA damaging agent and trigger for pattern recognition
receptors, respectively. Finally, this research will give insight into the evolution of LINE-1-like retroelements
that are found across all kingdoms of life.
Terms: <5' Capped RNA><5' mRNA Cap Structure><Aging><Aicardi Goutieres syndrome><Amino Acids><Assay><Autoimmune Diseases><Bioassay><Biochemical><Biologic Assays><Biological Assay><Bombyx mori><Breast Cancer><CREE ENCEPHALITIS><Cancer Biology><Cancers><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Chromosomal Rearrangement><Clinical><Code><Coding System><Colon Cancer><Colon Carcinoma><Cytoplasm><DNA><DNA Damage><DNA Damage Repair><DNA Injury><DNA Nucleotidylexotransferase><DNA Repair><DNA Sequence Rearrangement><DNA-Dependent RNA Polymerase II><Data><Degenerative Disorder><Deoxynucleotidyl Transferase><Deoxynucleotidyltransferase><Deoxyribonucleic Acid><Desoxynucleotidyl Transferase><Desoxynucleotidyltransferase><Disease><Disorder><Dissociation><EC 2.7.7.49><Encephalopathy with intracranial calcification and chronic lymphocytosis of cerebrospinal fluid><Encephalopathy, familial infantile, with calcification of basal ganglia and chronic cerebrospinal fluid lymphocytosis><Esophageal Cancer><Esophagus Cancer><Evolution><Gel><Gene Transcription><Genetic Transcription><Genome><Genomics><Goals><H-bond><Human><Human Genome><Hydrogen Bonding><IFN><Immune signaling><In Vitro><Interferons><Intervening Sequences><Intracellular Communication and Signaling><Introns><L1 Elements><LINE-1 Element><Label><Length><Lesion><Life><Life Cycle><Life Cycle Stages><Long Interspersed Elements><Lupus Erythematosus Disseminatus><Macular degeneration><Macular degenerative disease><Malignant Esophageal Neoplasm><Malignant Esophageal Tumor><Malignant Neoplasms><Malignant Tumor><Malignant Tumor of the Esophagus><Malignant neoplasm of esophagus><Measures><Mediating><Modern Man><Modification><Non-Polyadenylated RNA><Nucleic Acids><Nucleosides><Nucleotides><ORFs><Open Reading Frames><Pattern recognition receptor><Peptide Domain><Phenotype><Phylogenetic Analysis><Phylogenetics><Polymerase><Polymers><Process><Proliferating><Property><Protein Coding Region><Protein Domains><Proteins><Pseudouridine><RNA><RNA Binding><RNA Caps><RNA Expression><RNA Gene Products><RNA Polymerase B><RNA Polymerase II><RNA Processing><RNA Transcriptase><RNA bound><RNA vaccine><RNA-Binding Proteins><RNA-Dependent DNA Polymerase><RNA-Directed DNA Polymerase><RNA-based vaccine><Reaction><Reading><Research><Research Proposals><Resolution><Retroelements><Retrotransposition><Retrotransposon><Reverse Transcriptase><Reverse Transcriptase Inhibitors><Reverse Transcription><Revertase><Ribonucleic Acid><Ribonucleoside Phosphates><Ribonucleotides><Role><SINEs><SLE><Series><Short Interspersed DNA Sequence Elements><Short Interspersed Element><Short Interspersed Nucleotide Elements><Signal Transduction><Signal Transduction Systems><Signaling><Structure><Systemic Lupus Erythematosus><Systemic Lupus Erythematous><Systemic Lupus Erythmatosus><Terminal Addition Enzyme><Terminal Deoxynucleotidyl Transferase><Terminal Deoxynucleotidyltransferase><Terminal Deoxyribonucleotidyl Transferase><Terminal Deoxyribonucleotidyltransferase><Tertiary Protein Structure><Testing><Thumb><Thumb structure><Transcript><Transcription><Unscheduled DNA Synthesis><Work><aminoacid><autoimmune condition><autoimmune disorder><autoimmunity disease><biological signal transduction><cancer in the colon><clinical significance><clinically significant><degenerative condition><degenerative disease><disseminated lupus erythematosus><driving force><experiment><experimental research><experimental study><experiments><genomic rearrangement><human disease><human whole genome><improved><in vitro activity><insight><interest><knowledge base><life course><mRNA vaccine><mRNA-based vaccine><malignancy><malignant breast neoplasm><malignant breast tumor><microcephaly-chorioretinopathy syndrome><neoplasm/cancer><oesophageal cancer><polymer><polymeric><polymerization><pseudotoxoplasmosis syndrome><public health research><repair><repaired><resolutions><social role><systemic lupus erythematosis>