Erythroid Cell-Specific mRNA Stability Elements in the α2-Globin 39 Nontranslated Region

Abstract

Very little is known about the mechanisms mediating longevities of mRNAs. As a means of identifying potential cis-and trans-acting elements which stabilize an individual mRNA, naturally occurring mutations that decreased stability of the normally long-lived globin mRNA were analyzed. Our previous studies demonstrated that a subset of mutations which allowed the translating ribosome to read through into the ␣2-globin 3 nontranslated region (NTR) targeted the mutant mRNAs for accelerated turnover in erythroid cells but not in several nonerythroid cell lines (I. M. Weiss and S. A. Liebhaber, Mol. Cell. Biol. 14:8123-8132, 1994). These results suggested that translational readthrough interfered with some feature of the ␣2-globin 3 NTR required for message stability in erythroid cells. To define the cis-acting sequences which comprise this erythroid cell-specific stability determinant, scanning mutagenesis was performed on the ␣2-globin 3 NTR, and the stability of each mutant mRNA was examined during transient expression. Three cytidine-rich regions which are required for longevity of the ␣2-globin mRNA were identified. However, in contrast to the readthrough mutations, base substitutions in these elements destabilize the message through a translation-independent mechanism. To account for these results, we propose that the cis-acting elements form a complex or determinant in the normal ␣2-globin mRNA which protects the message from degradation in erythroid cells. Disruption of this determinant, by translational readthrough or because mutations in an element prevent or inhibit its formation, targets the message for accelerated turnover in these cells.

The poly(C)-binding proteins: A multiplicity of functions and a search for mechanisms

“…PCBPs have been implicated in a wide spectrum of posttranscriptional controls+ Initial insights into the roles emerged from studies of human a-globin mRNA stabilization (Weiss & Liebhaber, 1994+ Highlevel stability of a-globin mRNA is essential to full expression of globin proteins during the 2-3 days of transcriptionally silent terminal erythroid differentiation+ Stabilization of a-globin mRNA is tightly linked to formation of a binary complex between a single molecule of aCP and a pyrimidine-rich motif within the a-globin 39 UTR ("a-complex"; Fig+ 4A; Kiledjian et al+, 1995;Wang et al+, 1995;Chkheidze et al+, 1999)+ Interruption of any of the three C-rich patches within this region disrupts the a-complex assembly in vitro and decreases a-globin mRNA stability in vivo (Wang et al+, 1995;Weiss & Liebhaber 1995)+ aCP-1 and aCP-2 can independently bind to the human a-globin mRNA 39 UTR to form the a-complex as demonstrated in vitro+ This binding is specific to aCPs as hnRNP K lacks this binding activity (Chkheidze et al+, 1999)+ The respective K d values of recombinant aCP-1 (51 nM) and aCP-2 (0+8 nM) FIGURE 4. Examples of functions mediated by the PCBPs+ Six distinct examples of PCBP function are shown+ In each case, the PCBP isoform of interest is indicated as a filled circle+ A specific example is given for each of these indicated functions and each is expanded upon in the text+ The mRNAs are indicated in diagrammatic format with tick marks indicating the initiation and termination codons+ The cellular mRNAs are also shown with cap (filled circles) and poly(A) tails+ The polioviral mRNA lacks a cap structure; the two IRES structures of specific interest, secondary structures known as Domain I and Domain IV, are shown+ Question marks indicate unidentified protein factors and/or assumed protein-protein interactions (as discussed in the text)+ 272…”

Regulation of Tyrosine Hydroxylase mRNA Stability by Protein-binding, Pyrimidine-rich Sequence in the 3′-Untranslated Region

“…Two 1-5) or CAT containing either wild-type TH sequence (CAT-wtTH, lanes 6 -10) or mutated TH sequence (CAT-mutTH, lanes [11][12][13][14][15] at indicated times after inhibition of transcription with actinomycin D (AD). Lower panels show less exposed 18 S bands and indicate equal loading of RNA.…”

The Poly(A) Tail Inhibits the Assembly of a 3′-to-5′ Exonuclease in an In Vitro RNA Stability System

“…Several internal sequences, such as an AU-rich element (10) or nonsense codons (5), which functionally shorten the half-lives (t 1/2 ) of mRNAs have been identified. Furthermore, an internal element which stabilizes ␣ globin mRNA has recently been identified (38). Elucidating how the general and regulatory elements interact to determine the functional t 1/2 of mRNAs is vital to understanding the mechanism of RNA stability as a regulator of gene expression.…”