Downstream sequence elements with different affinities for the hnRNP H/H' protein influence the processing efficiency of mammalian polyadenylation signals

Abstract

Auxiliary factors likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We previously identified an auxiliary factor, hnRNP H/H', which stimulates 3'-end processing through an interaction with sequences downstream of the core elements of the SV40 late polyadenylation signal. Using in vitro reconstitution assays we have demonstrated that hnRNP H/H' can stimulate processing of two additional model polyadenylation signals by binding at similar relative downstream locations but with significantly different affinities. A short tract of G residues was determined to be a common property of all three hnRNP H/H' binding sites. A survey of mammalian polyadenylation signals identified potential G-rich hnRNP H/H' binding sites at similar downstream locations in approximately 34% of these signals. All of the novel G-rich elements tested were found to bind hnRNP H/H' protein and the processing of selected signals identified in the survey was stimulated by the protein both in vivo and in vitro. Downstream G-rich tracts, therefore, are a common auxiliary element in mammalian polyadenylation signals. Sequences capable of binding hnRNP H protein with varying affinities may play a role in determining the processing efficiency of a significant number of mammalian polyadenylation signals.

Figure 1

HnRNP H protein shows signal-specific effects on 3′-end processing. (A) The IVA₂, µM and SVL polyadenylation substrate RNAs were incubated in the in vitro 3′-end cleavage system in the absence (– lanes) or presence (+ lanes) of 500 ng recombinant hnRNP H protein. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The presence of hnRNP H increased the processing efficiency of the IVA₂, µM and SVL polyadenylation signals by 3.7 ± 0.8-, 3.9 ± 0.8- and 3.6 ± 0.5-fold, respectively. (B) The SVL polyadenylation substrate RNA was incubated in the in vitro 3′-end cleavage system in the presence of the indicated amounts of a synthetic competitor RNA, GRS, that can bind and sequester hnRNP H protein. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The processing efficiency relative to 0 pmol GRS RNA competitor is shown below each lane. (C) The IVA₂ polyadenylation substrate RNA was incubated in the in vitro 3′-end cleavage system in the presence of the indicated amounts of synthetic GRS competitor RNA. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The processing efficiency relative to 0 pmol GRS RNA competitor is shown below each lane. The arrows on the left of (B) and (C) indicate the positions of the 5′ cleavage product.

Figure 2

Differential binding affinity of hnRNP H affects its ability to stimulate 3′-end processing of the SVL and IVA₂ polyadenylation signals. SVL (A) and IVA₂ (B) RNAs were incubated with the indicated amount of recombinant hnRNP H protein. Heparin-resistant RNA–protein complexes were analyzed on 5% native acrylamide gels. The arrows on the right of each panel indicate the positions of hnRNP H–RNA complexes. (C) The indicated amounts of hnRNP H protein were added to in vitro cleavage reactions containing either the SVL or IVA₂ polyadenylation signal RNAs. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. hnRNP H at 15 ng had a 4.2-fold greater effect on the SVL versus the IVA₂ polyadenylation signal. hnRNP H at 100 ng stimulated both signals to a similar extent (6.8-fold).

Figure 3

hnRNP H protein binds to G-rich sequences located at a conserved downstream position in the SVL, IVA₂ and µM polyadenylation signals. (A) The IVA₂ polyadenylation substrate RNA or the indicated derivatives diagrammed at the top were incubated with 500 ng recombinant hnRNP H protein. Heparin-resistant RNA–protein complexes were analyzed on a 5% native acrylamide gel. The arrow on the left indicates the position of hnRNP H–RNA complexes. (B) The µM polyadenylation substrate RNA or the indicated derivatives diagrammed at the top were incubated with 500 ng recombinant hnRNP H protein. Heparin-resistant RNA–protein complexes were analyzed on a 5% native acrylamide gel. (C) Sequences required for hnRNP H binding by the IVA₂ and µM polyadenylation signals. Conserved G-rich regions that may be required for binding are underlined.

Figure 4

The downstream hnRNP H binding site is required for efficient processing and stimulation of the IVA₂ polyadenylation signal. (A) Diagrammatic representation of the RNAs used in this study. (B) The IVA₂ polyadenylation substrate RNA or IVA₂-GEM, a derivative that contains a substitution in the hnRNP H binding region, were incubated with 500 ng recombinant hnRNP H protein. Heparin-resistant RNA–protein complexes were analyzed on a 5% native acrylamide gel. The arrow on the right indicates the position of hnRNP H–RNA complexes. (C) IVA₂ or IVA₂-GEM RNAs were incubated in the in vitro 3′-end cleavage system in the absence (no protein lanes) or presence (hnRNP H lanes) of 1 µg recombinant hnRNP H protein. Reaction products were analyzed on a 5% acrylamide gel containing 7 M urea. The arrow on the left indicates the position of the 5′ cleavage product. The addition of hnRNP H to the IVA₂ polyadenylation signal resulted in a 6.8-fold increase in processing efficiency in this assay, while the protein had no effect on processing of the IVA₂-GEM RNA substrate.

Figure 5

G-rich regions located in the downstream region of ∼34% of polyadenylation signals surveyed are capable of directing the binding of hnRNP H protein. Small RNAs containing the G-rich downstream region from the indicated polyadenylation signal (Table 1) were incubated with 1 µg recombinant hnRNP H protein. Heparin-resistant RNA–protein complexes were analyzed on a native 5% acrylamide gel.

Figure 6

Processing of the cellular RATCRP2A polyadenylation signal is stimulated by hnRNP H protein. Substrate RNA containing the SVL or RATCRP2A polyadenylation signal was incubated in the in vitro polyadenylation system in the absence (– lane) or presence (+ lane) of 250 ng recombinant hnRNP H protein . Reaction products were identified on a 5% acrylamide gel containing 7 M urea. The positions of input and polyadenylated RNA products of the reaction are indicated on the left. The addition of hnRNP H protein to the reaction stimulated processing by 4.1 ± 0.8-fold.

Figure 7

Expression of transcripts containing G-rich sequences downstream of their polyadenylation/cleavage sites is stimulated in hnRNP H′-transfected cells. AxJ plasma cells were transfected with an expresssion vector carrying either no insert (none lanes), hnRNP H′ coding sequence (lanes H′) or hnRNP F coding sequences (lanes F). Poly(A)⁺ RNA (1 µg) was run on a denaturing formaldehyde gel, blotted to membrane and probed for the indicated endogenous mouse mRNAs. (A) MUSPKCD10 gene (organization of the polyadenylation signal is outlined in Table 1). (B) Lane 1, cells transfected with vector containing no insert; lanes 2 and 3, vector plus hnRNP H′ coding region; lanes 4 and 5, vector plus hnRNP F coding region. Blots were probed for the mRNA indicated on the right. (Bottom) Organization of the AAUAAA motif, U-rich CstF-binding site and G-rich elements in the mouse Gpi1 and Psmb5 polyadenylation signals. Numbers indicate the position relative to the CA or GA cleavage site. A consensus CstF-binding site could not be identified in the downstream region of the Psmb5 signal.