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. 2022 Feb 5;13(2):305.
doi: 10.3390/genes13020305.

The Ribosomal Protein RpL22 Interacts In Vitro with 5'-UTR Sequences Found in Some Drosophila melanogaster Transposons

Crescenzio Francesco Minervini  1 , Maria Francesca Berloco  2 , René Massimiliano Marsano  2   3 , Luigi Viggiano  2
Affiliations

The Ribosomal Protein RpL22 Interacts In Vitro with 5'-UTR Sequences Found in Some Drosophila melanogaster Transposons

Crescenzio Francesco Minervini et al. Genes (Basel). .

Abstract

Mobility of eukaryotic transposable elements (TEs) are finely regulated to avoid an excessive mutational load caused by their movement. The transposition of retrotransposons is usually regulated through the interaction of host- and TE-encoded proteins, with non-coding regions (LTR and 5'-UTR) of the transposon. Examples of new potent cis-acting sequences, identified and characterized in the non-coding regions of retrotransposons, include the insulator of gypsy and Idefix, and the enhancer of ZAM of Drosophila melanogaster. Recently we have shown that in the 5'-UTR of the LTR-retrotransposon ZAM there is a sequence structured in tandem-repeat capable of operating as an insulator both in Drosophila (S2R+) and human cells (HEK293). Here, we test the hypothesis that tandem repeated 5'-UTR of a different LTR-retrotransposon could accommodate similar regulatory elements. The comparison of the 5'-UTR of some LTR-transposons allowed us to identify a shared motif of 13 bp, called Transposable Element Redundant Motif (TERM). Surprisingly, we demonstrated, by Yeast One-Hybrid assay, that TERM interacts with the D. melanogaster ribosomal protein RpL22. The Drosophila RpL22 has additional Ala-, Lys- and Pro-rich sequences at the amino terminus, which resembles the carboxy-terminal portion of histone H1 and histone H5. For this reason, it has been hypothesized that RpL22 might have two functions, namely the role in organizing the ribosome, and a potential regulatory role involving DNA-binding similar to histone H1, which represses transcription in Drosophila. In this paper, we show, by two independent sets of experiments, that DmRpL22 is able to directly and specifically bind DNA of Drosophila melanogaster.

Keywords: DNA-protein interaction; Drosophila; Rpl22; histone 1-like; ribosomal protein; transposable elements.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Full-length RpL22 cDNA clone. Graphical representation of the RpL22 cDNA clone which was used to construct vectors expressing RpL22 and its sub-domains (ribosomal and histone-like). Arrows indicate the name and position of the PCR oligo-primers.
Figure 2
Figure 2
Bioinformatics analysis of the 5′-UTRs of ZAM, Tirant, and Accord. (A) Feature map of over-represented TERM in the 5-UTRs of the indicated RTs. The scale bar provides coordinates relative to the first ORF (GAG) start of the retrotransposons. Note the regularity of the TERM motif in the 5′-UTRs; (B) display of the logos of the TERM motif. The graphic representation was created using WebLogo. Sequence logos are a graphical representation of an alignment of multiple nucleic acid sequences (PWM) developed by Tom Schneider and Mike Stephens [34]. Each logo is made up of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the conservation of the sequence at that location, while the height of the symbols within the stack indicates the relative frequency of each nucleic acid at that location; (C) positional weight Matrix of TERM motif; (D) sequence of the tandem repeats present in the 5′-UTR of the RTEs under examination. The single tandem repeats are in blue and red, while the TERM motifs are in uppercase underscored.
Figure 3
Figure 3
Rpl22 binds the TERM3 in vitro. Each lane contains an identical amount of input labeled TERM3 DNA (2 ng) incubated with recombinant purified Rpl22 protein. (A) TERM3-Rpl22 complex formation has shown in the lane 2, whereas the remaining lanes are committed to specific and not-specific competition experiments: specific competitor (unlabeled TERM3*) or a large excess of non-specific competitor (shared λ-DNA) were used as shown in figure; (B) identification of which domain of RpL22 is responsible for binding with TERM3: we used purified Rpl22 (1.8 µg), RpL22/H5 (1.2 µg), and RpL22/L22 (0.6 µg). We used different amounts of the proteins to maintain the same stoichiometric ratio. The experiment suggests that only the RpL22/H5 polypeptide is able to bind TERM3.
Figure 4
Figure 4
RpL22 localization in Drosophila cell line S2R+ and in the brain cells. (A) Rpl22 localizes both in cytoplasm and nucleolus in S2R+ cell. (B) To highlight the “ribosomal” behavior of RpL22, co-immunofluorescence experiments were performed both with the anti-H1 antibody and anti-RpL28 antibody, finally, as further confirmation of the nucleolar localization, RpL22 co-localizes with the nucleolar marker of fibrillarin. (C) The same localization pattern occurs (cytoplasm and nucleolus) also in neurons.
Figure 5
Figure 5
Comparison of the expression profile of RpL22 (in blue) with the other ribosomal proteins during the development of Drosophila melanogaster. Microarray data of ribosomal D. melanogaster gene expression during development was downloaded from the FLYMINE database [49] (available at: https://www.flymine.org/flymine; last accessed 15 December 2021). These data were used to construct the graph. Y axis: fold change. Reference sample is a pooled mRNA representing all stages of the life cycle as reported in Arbeitman et al. [48].
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