A human Alu RNA-binding protein whose expression is associated with accumulation of small cytoplasmic Alu RNA.

Abstract

Human Alu sequences are short interspersed DNA elements which have been greatly amplified by retrotransposition. Although initially derived from the 7SL RNA component of signal recognition particle (SRP), the Alu sequence has evolved into a dominant transposon while retaining a specific secondary structure found in 7SL RNA. We previously characterized a set of Alu sequences which are expressed as small cytoplasmic RNAs and isolated a protein that binds to these transcripts. Here we report that biochemical purification of this protein revealed it as the human homolog of the SRP 14 polypeptide which binds the Alu-homologous region of 7SL RNA. The human cDNA predicts an alanine-rich C-terminal tail translated from a trinucleotide repeat not found in the rodent homolog, which accounts for why the human protein-RNA complex migrates more slowly than its rodent counterpart in RNA mobility shift assays. The human Alu RNA-binding protein (RBP) is expressed after transfection of this cDNA into mouse cells. Expression of human RBP in rodent x human somatic cell hybrids is associated with substantial increase in endogenous small cytoplasmic Alu and scBl transcripts but not other small RNAs. These studies provide evidence that this RBP associates with Alu transcripts in vivo and affects their metabolism and suggests a role for Alu transcripts in translation in an SRP-like manner. Analysis of hybrid lines indicated that the Ahu RBP gene maps to human chromosome 15q22, which was confirmed by Southern blotting. The possibility that the primate-specific structure of this protein may have contributed to Alu evolution is considered.Alu sequences are short transposed elements endogenous to the human genome (24, 55). These elements have accumulated to nearly 1 million copies comprising approximately 5% of human DNA. Alu repeats have sequence homology with and are believed to have been derived from the Alu-homologous region of the 7SL RNA component of signal recognition particle (SRP) (64,69,70,71). The current model of Alu evolution suggests that two independently derived Alu-homologous domains of 7SL dimerized to form the more efficient transposing element known as Alu (26,28,45,46,47). Since then, Alu repeats have continued to evolve within primate genomes. A major advance in understanding Alu mobility was the result of work by researchers in several laboratories who classified Alu repeats into subfamilies according to their evolutionary relatedness as determined by sequence homology (8,27,45,59,72). This, together with recent phylogenetic evidence, indicates that multiple Alu source genes continue to spawn new transposed elements (22, 25, 31, 39, 42, 67; reviewed in reference 49).Why genetic elements with disruptive potential have been allowed to proliferate to high copy number in human DNA remains enigmatic. Perhaps Alu mobility improves adaptability of the host genome, or perhaps Alu DNA elements or their RNA products have adopted a function(s) which more directly benefits their host cells (9,19,51,74). Given their short lengt...

The Decline in Human Alu Retroposition Was Accompanied by an Asymmetric Decrease in SRP9/14 Binding to Dimeric Alu RNA and Increased Expression of Small Cytoplasmic Alu RNA

“…For in vitro RNA synthesis, promoters for T7 RNA polymerase were positioned by PCR to initiate RNA synthesis at the first base of the Alu sequence and to terminate at the last base of the Alu sequence as described previously (30). Alu monomer electrophoretic mobility shift assays (EMSAs) were performed with SRP9/14 that was purified from HeLa cells (heparin agarose fraction) (10). Alu dimer EMSAs were performed with cytoplasmic extract of owl monkey cells as described previously (11,19).…”

“…The resulting Alu DNAs were cloned into the HindIII/EcoRI sites of pUC18 (Bethesda Research Laboratories) and designated pAlu-PS (Sx), pAlu-CS (Y), and pAlu-HS (Ya5). Transfection was carried out as described previously (10). RNA purification and denaturing PAGE were as described previously (30) except that the DNase I treatment was for 2 h, as we found this necessary to fully clear the transfected plasmid.…”

“…These regions in 7SL RNA base pair to form a specific cruciform structure that is recognized by the heterodimeric SRP protein known as SRP9/14 (18,49). This structure is grossly conserved in consensus flAlu RNAs, and it has been shown that cellular scAlu transcripts associate with SRP9/14 (5,10,25,31,42). Conservation of the Alu cruciform structure by flAlu consensuses suggests that interaction with SRP9/14 has had a positive effect on Alu retroposition, presumably at the level of RNA stability.…”

Crystallization and preliminary crystallographic analysis of the signal recognition particle SRPΦ14‐9 fusion protein

“…Targeting occurs co-translationally and translocation across the RER membrane begins before polypeptide synthesis is complete [3][4][5]. The SRP acts in three distinct ways [6]: it binds the signal sequence of the nascent polypeptide to be translocated [4,6,7], which is exposed on the surface of the translating ribosome [8,9]; it temporarily retards the nascent polypeptide from further elongation [6,10,11]; and it mediates docking of the SRP-ribosome-nascent polypeptide chain complex to the RER membrane via the heterodimeric SRP receptor. With the engagement of this machinery, the SRP is detached and recycled, and co-translational transloca-*Corresponding author.…”

“…It is possible that SRP9/14 might possess a new RNA binding motif which may be generated upon heterodimerization since neither protein alone binds specifically to SRP RNA. Recently, it has been shown that human SRP9/14 in vivo [7], and SRP14 in vitro [25] can associate as complexes with scAlu RNA in so-called Alu particles. Furthermore, the human SRP14 is larger than its murine counterpart, this difference being due to a C-terminal alanine/threonine-rich extension [25].…”