3' end mRNA processing: molecular mechanisms and implications for health and disease

Abstract

Recent advances in the understanding of the molecular mechanism of mRNA 3' end processing have uncovered a previously unanticipated integrated network of transcriptional and RNA-processing mechanisms. A variety of human diseases impressively reflect the importance of the precision of the complex 3' end-processing machinery and gene specific deregulation of 3' end processing can result from mutations of RNA sequence elements that bind key specific processing factors. Interestingly, more general deregulation of 3' end processing can be caused either by mutations of these processing factors or by the disturbance of the well-coordinated equilibrium between these factors. From a medical perspective, both loss of function and gain of function can be functionally relevant, and an increasing number of different disease entities exemplifies that inappropriate 3' end formation of human mRNAs can have a tremendous impact on health and disease. Here, we review the mechanistic hallmarks of mRNA 3' end processing, highlight the medical relevance of deregulation of this important step of mRNA maturation and illustrate the implications for diagnostic and therapeutic strategies.

Figure 1

Cis-acting sequence elements and trans-acting factors involved in mammalian 3′ end processing. (A) 3′ end processing of polyadenylated mRNAs. After assembly of multiprotein complexes at the respective RNA recognition motifs (upper panel), the primary transcript is endonucleolytically cleaved at the cleavage site by CPSF 73. This is followed by the addition of adenine residues to the 3′ end to form a poly(A) tail that is bound by PABPN1. The interaction of PAP with this protein and with CPSF is critical to establish the processive action of the polymerase for the synthesis of approximately 250 A residues. Following polyadenylation, the interaction of PABPN1 with the poly(A) tail is characterized by a rapid on–off rate, and PABN1 is exchanged by cytoplasmic PAPB (PABPC) at the time of nuclear export. PABPC interacts with the translation initiation factor elF4G as part of the initiation complex thus generating a translation competent pseudocircular ribonucleoprotein particle. (B) In contrast to mRNAs with a poly(A) site, 3′ end processing of replication-dependent histone mRNAs requires conserved sequence elements and structural elements. The signal for 3′ end processing of this small class of pre-mRNAs consists of a conserved stem–loop element that is positioned upstream of the cleavage site and a purine-rich HDE. The HDE is recognized by base pairing with the 5′ end of the U7 small nuclear RNA, which is incorporated into a U7 snRNP. The stem–loop is bound by the SLBP, which functions in 3′ end processing, translation and in the coupling of message stability to DNA replication and the cell cycle. After recruitment of the 3′ end-processing apparatus, the site of endonucleolytic cleavage occurs 3′ of a CA dinucleotide 9–12 nucleotides upstream of the intermolecular U7/HDE RNA duplex. The SLBP also serves to establish a pseudo-circularization together with initiation factor elF4G. Stimulatory interactions are highlighted (+). Cis-acting RNA elements and trans-acting factors are summarized inTables I and II, USE=upstream sequence element, AAUAAA=poly(A) signal, DSE=downstream sequence element, HDE=Histone downstream element, CstF=cleavage stimulating factor (blue complex), CPSF=cleavage/polyadenylation specificity factor (light blue complex), Pol II=RNA polymerase II (light green complex) with CTD (C-terminal domain), CF I=cleavage factor I (green complex), CF II=cleavage factor II (grey complex), PAP=poly(A)-polymerase (yellow complex), PABPN1=nuclear poly(A)-binding protein, PABPC=cytoplasmic poly(A)-binding protein (dark blue complex), 4A, 4E, 4G=translation initiation factors (grey complex), SLBP=stem loop binding protein (yellow complex).

Figure 2

Integrated networks of co-transcriptional mRNA processing to regulate gene expression and to maintain genomic stability. (A) Transcription initiation, elongation and termination (circular arrow) are tightly coupled to mRNA processing steps such as capping, splicing and 3′ end processing (inner circle). Appropriate 3′ end processing is functionally interconnected with transcription and mRNA capping and splicing, and impacts on post-transcriptional mechanisms (mRNA release, export, abundance and translation). Loss or gain of function of 3′ end processing thus critically interferes with other gene expression steps. (B) Co-transcriptional mRNA processing is believed to promote packaging of the nascent RNA transcript (formation of an ‘inert' RNP particle, upper panel) and thus to prevent the accumulation of co-transcriptional R-loops (lower panel), which can lead to DNA double strand breaks and chromosomal rearrangements. Disruption of co-transcriptional RNA processing is therefore thought to result in genomic instability (for review see Li and Manley, 2006).

Figure 3

The human prothrombin 3′ end-processing signal shows an unusual architecture of non-canonical sequence elements, which are susceptible to clinically relevant gain-of-function mutations. A sequence comparison of the 3′ end-processing signals of efficiently processed mRNAs such as SV40 late or β-globin (HBB) with F2 (lower lane) revealed an inefficient F2 cleavage dinucleotide context and a uridine-poor DSE. In contrast, the F2 3′ untranslated region contains a uridine-rich USE that promotes 3′ end processing and hence balances F2 mRNA expression. Sequences encompassing the cleavage site and the 3′-flanking region represent a vulnerable region for clinically relevant gain-of-function mutations.

Figure 4

Regulated and alternative 3′ end processing in development and disease. (A) During B-cell differentiation, alternative poly(A) site selection effects a switch of the IgM heavy-chain expression from a membrane-bound form (μm) to the secreted form (μs). In this example, CstF-64 binding to the site of the RNA giving rise to secreted IgM (μs) is favored either by high CstF-64 concentrations (Takagaki et al, 1996) or under conditions of low hnRNP F and/or low U1A concentrations (Veraldi et al, 2001; Phillips et al, 2004) in plasma cells (lower lane). In contrast, the high-affinity site of the membrane bound form (μm) is used in B cells (upper lane), where the CstF-64 concentration is low or when high concentrations of U1A and/or hnRNP F inhibit CstF-64 binding to the secretory μs-specific poly(A) site (modified after Barabino and Keller, 1999; boxes indicate exons). (B) The BRCA1-associated protein BARD1 physically interacts with CstF-50, thereby repressing the polyadenylation machinery (Kleiman and Manley, 1999). Both BARD1 and CstF-50 also interact with Pol II (not shown), and BARD1 has been proposed to sense sites of DNA damage and repair. The BARD1-mediated inhibition of polyadenylation thus prevents inappropriate RNA processing during transcription at such compromised sites. Consequently, challenging cells with DNA-damaging agents results in a transient inhibition of 3′ end formation by enhanced formation of a CstF/BARD1/BRCA1 complex. Furthermore, a tumor-associated germline mutation in BARD1 (Gln564His) decreases its affinity to CstF-50 and renders the protein inactive in polyadenylation inhibition. These findings link 3′ end RNA processing with DNA repair, and loss of wild-type BARD1 could therefore lead to defective control of gene expression as a result of inappropriate polyadenylation (Kleiman and Manley, 2001). (C) In influenza A virus-infected cells, the highly abundant NS1 protein interacts with the cellular 30-kDa subunit of CPSF (Nemeroff et al, 1998) and PABPN1 (not shown) (Chen et al, 1999). This prevents binding of the CPSF complex to its RNA substrates and selectively inhibits 3′ end processing and nuclear export of host pre-mRNAs (adopted from; Nemeroff et al, 1998). In contrast, the 3′ terminal poly(A) sequence on viral mRNAs is produced by the viral transcriptase, which reiteratively copies a stretch of 4–7 uridines in the virion RNA templates. In addition, an endonuclease intrinsic to the viral polymerase cleaves cellular capped RNAs to generate capped fragments that serve as primers for the viral mRNA synthesis (so-called ‘cap-snatching mechanism'; Rao et al, 2003). Thus, by interfering with the activity of an essential 3′ end-processing factor, influenza has devised an efficient way to specifically shut off cellular gene expression and to facilitate viral gene expression (further detail see Nemeroff et al, 1998; Chen et al, 1999; Rao et al, 2003).

Figure 5

Regulated and alternative 3′ end processing modulates the temporal and spatial diversity of gene expression. About half of the human pre-mRNAs contain (multiple) alternative poly(A) signals. A large number of these pre-mRNAs has alternative, mostly tandem, arrays of poly(A) sites within the 3′ UTR. A smaller set of pre-mRNAs bears alternative poly(A) signals within intronic or exonic regions. In both the cases, endogenous and exogenous factors can modulate pre-mRNA poly(A) site selection by interfering with constitutive and/or auxiliary 3′ end-processing factors/subunits (upper panel; depicted are various scenarios on a single pre-mRNA). This results in various polyadenylated mRNAs that either code for identical (tandem terminal poly(A) sites) or C-terminally modified (internal poly(A) sites) proteins (see also Figure 4A). Furthermore, alternatively 3′ end-processed mRNAs can display different 3′ UTR properties (middle and lower panel; mRNA variants 1 and 2, respectively). This diversity can affect mRNA abundance, mRNA localization, mRNA transport and translation of the respective mRNA variant (lower lane). Importantly, interaction of AU-rich elements (AREs) with the respective binding proteins (ABP) has a dual function and can result in both mRNA stabilization and destabilization. Mutually exclusive binding of different trans-acting factors allows a complex modulation of different processing activities. Auxiliary 3′ end-processing sequences (USE) may represent target sites for trans-acting factors that modulate 3′ end formation efficiencies.