# Deciphering functions of the LINE-1 ORF2p protein

> **NIH NIH F31** · TULANE UNIVERSITY OF LOUISIANA · 2024 · $42,574

## Abstract

PROJECT SUMMARY
Approximately half of many mammalian genomes are comprised of repetitive sequences, which include Long
INterspersed Element-1 (L1). L1 utilizes its ORF2p protein to increase its copy number through a process known
as retrotransposition. Despite the abundance of L1 copies in their host genome, many steps in their
retrotransposition cycle including the exact functions of ORF2p and how ORF2p accesses chromatin for
integration are unknown. The annotated half of ORF2p includes the functional endonuclease (EN) and reverse
transcriptase (RT), which are required for retrotransposition. The unannotated half of ORF2p, which includes the
Cryptic region and C-terminus, are evolutionarily conserved among L1s from diverse species but lack homology
to other cellular proteins. The C-terminus is required for retrotransposition, but it has no known function. Progress
in understanding mechanisms underlying ORF2p functions in retrotransposition is impeded by the lack of an
ORF2p protein structure and the unknown functions of ORF2p Cryptic and C-terminus regions. Our lab also
discovered that L1 sequences contain multiple splice sites, which can impact their host through generation of
splice variants with cellular genes. However, how often the thousands of L1 inserts form chimeric transcripts with
host genes and how their incorporation influences the cellular transcriptome is unknown. Thus, there is a need
for comprehensive analysis of L1 contribution to splice variants in different species.
Our lab has generated novel genetic approaches to study the function of the unannotated ORF2p regions in
retrotransposition. We previously identified the Cryptic region of ORF2p and proposed for it to be a structural
anchor for EN and RT. Studies, including our own, have shown that both Cryptic and C-terminus are essential
for retrotransposition. Our preliminary data show that the C-terminus (1) interacts with the proposed structural
anchor, Cryptic and (2) interacts with histones. Our preliminary data also show that the L1 3′-end sequence is
incorporated into cellular splice variants through alternative splicing events. Based on these data, we
hypothesize that the C-terminus of ORF2p is essential for retrotransposition through its intra- and intermolecular
interactions and the incorporation of L1 3′end sequence into splice variants impacts the cellular transcriptome.
We propose two aims to evaluate these central hypotheses. Aim 1 investigates the sequence requirements
involved in C-terminus interactions with Cryptic and core histones and retrotransposition steps requiring these
interactions. Aim 2 investigates L1 3′-end sequence incorporation into chimeric splice variants and its impact on
gene expression. Our results will determine (1) functional role(s) of unannotated regions within ORF2p and (2)
an impact of L1 3′-end sequence on cellular transcriptome. Our results will generate a database of chimeric
transcripts containing L1 3′-end sequences in different spec...

## Key facts

- **NIH application ID:** 10901291
- **Project number:** 1F31GM154468-01
- **Recipient organization:** TULANE UNIVERSITY OF LOUISIANA
- **Principal Investigator:** Afzaal Shareef
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $42,574
- **Award type:** 1
- **Project period:** 2024-03-28 → 2025-03-27

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10901291

## Citation

> US National Institutes of Health, RePORTER application 10901291, Deciphering functions of the LINE-1 ORF2p protein (1F31GM154468-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10901291. Licensed CC0.

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