Introduction Ribosomal Frameshifting (PRF). PRF is commonly utilized in

            Introduction

ASXL genes encode regulatory proteins of the enhancer
of trithorax and polycomb (ETP) group, which regulate the expression of
homeotic genes during embryogenesis. The ASXL genes include ASXL1-3 and a
single homologous gene (ASX) in Drosophila. ASXL proteins act as epigenetic
scaffolds and bind to several transcription factors (Dinan, 2017). Translation
is the process when code from mRNA is decoded resulting in a specific amino
acid sequence. Translational recoding are exceptions to genetic coding where
sequences that program elongating in ribosomes shift the translational reading
frame and therefore alter the codons. Examples of this include Programed
Ribosomal Frameshifting (PRF). PRF is commonly utilized in virus
gene expression, where it serves to control the ratio of different enzymatic or
structural proteins, or to allow access to overlapping open reading frames
(ORFs) which increases the coding capacity of small virus genomes. The gene for
antizyme utilizes +1 PRF to regulate synthesis of antizyme as part of a
feedback loop where the efficiency of frameshifting increases in response to elevated
polyamine levels (Dinan, 2017). Identifying PRF sites is most practical in
cases where the sequence of the frameshift site is phylogenetically conserved;
and where ribosomes which shift frame do not immediately encounter a stop
codon, but synthesize a transframe protein product. A highly-conserved instance
of the influenza A virus UCC_UUU_CGU +1 shift site was identified. This is in a
central region of the ASXL1 coding sequence, and corresponds with a long +1
frame overlapping ORF and enhanced synonymous site conservation in the
zero-frame. A corresponding +1 frame overlapping ORF is also present in ASXL2, the
ORF is associated with a highly conserved arterivirus RG_GUC_UCU ?2 shift site (Dinan,
2017).

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Hypothesis

The hypothesis of this article is that ASXL gain-of-function
truncation mutants have defective and dysregulated forms of a natural ribosomal
frameshifting product.

            Methods

            The initial set of 37,257 human mRNA RefSeqs was
downloaded from NCBI, and sequences from the transcriptome shotgun assembly database
were added. Nucleotide sequences for each gene were retrieved from NCBI. The
ASXL2 gene predictions of many reptiles and birds have large N-terminal
deletions but were omitted because a minimum query coverage threshold of 80%
was set. Analysis of sequences shows that all contain conserved copies of the
frameshift site and large TF ORFs in the expected frames. For each of ASXL1 and
ASXL2, full-length zero-frame coding nucleotide sequences were translated,
aligned as amino acids with MUSCLE, and the amino acid alignments were used for
codon-based nucleotide alignments using EMBOSS tranalign. The zeroframe
sequences of all mRNAs were scanned for +1 or ?2 PRF sites. Sequence alignments
were analyzed for synonymous site conservation using synplot2 with amino acid
PhyML guide trees, alignments were mapped to reference sequence coordinates. Subsets
of sequences were selected for sequence logos and were created using WebLogo.

The Predictor of Natural Disordered Regions was used to predict disordered
regions within the ASXL and ASXL-TF proteins. To search for potentially stable
RNA structures adjacent to putative frameshift sites, the 120-nt regions
downstream of all putative shift sites were aligned using Clustal Omega, and
structures were predicted using RNAalifold. Pseudoknots were scanned for using
PKNOTS. RiboSeq datasets were retrieved from the NCBI short reads archive and
mapped to human rRNA, then to the ASXL1 and ASXL2 transcripts. Ribosome
footprint densities were calculated for the regions upstream and downstream of
the TF ORF and were mapped to this region of the 5? end coordinate with a +12
nt offset. To identify ASXL1 TF-region indels everything from 330 nt upstream
of the TF ORF to 74 nt downstream of the TF ORF was searched for against all
post-rRNA subtraction sequencing reads and ran through blast. The wildtype and
mutant sequences at each site were used to extract and count the number of raw
reads containing the wildtype or mutant sequences.

            Results

            There is a conserved overlapping ORF in a central
region of mammalian ASXL1 and ASXL2. Each transcript comprises 13 exons, with
exon 13 being the longest. The zero-frame coding regions are 1541, 1435 and
2248 codons respectively for each transcript. The conserved +1 and ?2 PRF shift
sites are UCC_UUU_CGU for ASXL1 and G_GUC_UCU for ASXL2. Ribosomes which
frameshift translate a conserved +1 frame ORF. It was also determined that
there is conservation of the frameshift site and overlapping ORF. Codon
alignments of selected ASXL sequences near the predicted frameshift sites. In
the case of ASXL1, the sequence was found to be conserved in each species for
which a sequence was identified, except the Australian ghostshark. Presence of
a CGG codon at this position reduces the PRF efficiency by ~50% which is consistent
with the occurrence of PRF in the ASXL1 gene of the Australian ghostshark. In
the case of ASXL2 the sequence was found to be conserved in all taxa except
lizards and teleost fish. In each alignment, highly significant synonymous site
conservation was observed in a region coincident with the +1 frame TF ORF. The
predicted PRF shift sites occur at the 5? end of the region of conservation and
often correspond to specific conservation peaks. Overlapping non-coding
features may lead to enhanced synonymous site conservation. The presence of a
long open reading frame in both ASXL1 and ASXL2 indicates an overlapping coding
sequence. Several transcript isoforms have been identified for ASXL1 and ASXL2.

Alignment of the zero-frame sequences of ASXL1 and ASXL2 suggests that the
splice junctions have been mis-annotated due to missing or incomplete
underlying genomic sequence data. Independent expression of the ASXL1 and ASXL2
TF polypeptides is unlikely given the lack of appropriately positioned AUG
codons within the TF ORF sequences. For both ASXL1 and ASXL2, conservation of
amino acids was observed in the N-terminal region of TF. It is evident that
amino acids with physicochemical properties are maintained at specific sites. The
core EHN/SY sequence matches the metazoan Host Cell Factor-1 binding motif, and
was the most significant match for the entire TF peptide sequence. Structural
analyses showed no significant homology between the TF peptide sequences and
tertiary structural domains, suggesting that the TF peptides do not harbor
functional tertiary structures, but exert a regulatory impact through conserved
short linear motifs. Most datasets did not show drop-off at the end of the TF
ORF for either ASXL1 or ASXL2. Jurkat ASXL1 ribosome drop-off supports
efficient PRF in certain cell types. Analysis of Gawron et al. RiboSeq reads revealed
two indel mutations, the first mimics a ?1 frameshift leading to premature
termination in the middle of the TF ORF, and and the second mimics a +1
frameshift leading to termination at the TF stop codon. Current ribosome
profiling datasets neither support nor contradict the PRF hypothesis. It is
likely that ASXL PRF would not be detectable by this approach.

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