Differentiation (1981) 18: 125-131 Differentiation 0 Springcr-Verlag 1981

Models and Hypotheses

A Possible Regulatory Mechanism in RNA Processing and Its Implication for Posttranscriptional Sequence Control during Differentiation of Cell Function H. NAORA and N. J. DEACON Molecular Biology Unit, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra, A.C.T. 2601, Australia

1. This paper is concerned with the possible molecular mechanism for RNA processing including posttranscriptional sequence control underlying the differentiation of cell func- tions.

2. It was previously postulated that intramolecular double-stranded hairpin structures present at 5‘- and 3‘-terminal regions of a ‘pre-mRNA’ are key elements for RNA splicing

3. In this paper the possibility is considered that the splicing of ‘pre-mRNA’ can be regulated in such a way that the formation of the proper double-stranded hairpin structures is prevented by the binding of low-molecular-weight nuclear RNA (LnRNA) to the terminal regions and/or to the nucleotide sequences around the exon-intron and intron-exon joint sites of the ‘pre-mRNA’ molecules.

4. Complementarity assessment of nucleotide sequences of rat preproinsulin ‘pre-mRNA’ and rat LnRNA, i.e. U1, showed that U1 RNA is capable of forming stable double-stranded intermolecular structures around the joint sites of preproinsulin ‘pre-mRNA’ and may prevent the formation of intramolecular double-stranded structures required for FUUA splicing. This may imply a regulatory (inhibitory) role for U1 RNA in the processing of ‘pre-mRNA’.

5. A possible regulatory role of LnRNA in RNA splicing is discussed in relation to the determination of the mRNA population to be translated in the cytoplasm during differentiation of cell functions.

~ 4 1 .

1. Introduction

There is considerable evidence to demonstrate that a mechanism which involves the switching on or off of gene transcription operates as a key element in the differentiation of cell functions [l]. Recent experi- ments have shown that a posttranscriptional sequence selection also contributes significantly to the deter- mination of mRNA populations to be translated in the cytoplasm during differentiation of cell functions and virus infection [2- 121 via elaborate processing pathways, including intron removal and exon splicing [13, 141. This paper is concerned with the possible molecular mechanism for RNA processing, including

posttranscriptional sequence control, underlying such differentia tion.

2. Prerequisite Data

Double-stranded structures are thought to be involved in the processing of large RNA transcripts [15-20, reviewed in 14, 211. Intramolecular dou- ble-stranded hairpin structures have been demon- strated for both 5’- and 3‘-terminal regions of a pre-mRNA molecule [21-241. Recent studies on nucleotide sequence complementarity of some pre- sumptive ‘pre-mRNAs’ have shown that nucleotide

0301-4681/81/0018/0125/$ 01.40

126 H. Naora and N. J. Deacon: RNA Processing Mechanism

sequences at the 5’- and 3’-terminal regions of these molecules are complementary to those at the exon-in- tron and intron-exon joint sites of the same ‘pre-mRNA’ molecules [24]. Based on these findings, we have previously proposed a model of RNA splicing: the 5’- and 3’-terminal nucleotide sequences involved in the double-stranded hairpin structures serve as the basic structural element to allow two splicing sites to stand close together, and thus splicing takes place at these specific regions of the pre-mRNA molecules without any intermolecular aid from other special RNA sequences [24].

An interesting prediction from this model would be that the splicing of pre-mRNA could be regulated in such a way that the formation of the proper double-stranded hairpin structures is prevented, e.g. , by binding of a regulatory RNA to the 5’- and 3’-terminal regions and/or to the nucleotide sequences around the exon-intron and intron-exon joint sites of the pre-mRNA molecules. A series of recent experiments on the nature of low-molecu- lar-weight nuclear RNA (LnRNA) [25, 261 has suggested the possible involvement of LnRNA in RNA processing. Firstly, ribonucleoprotein particles containing pre-mRNA possess LnRNA or are func- tionally associated with the structures containing LnRNA [27-321. Secondly, LnRNA is indeed asso- ciated with or can be hybridized to poly(A) contain- ing nuclear RNA [33-35, and N. T. Patel, and V. Holoubek, in preparation]. Thirdly, the nucleotide sequences of some LnRNA or RNA, presumably transcribed from repeated nucleotide sequences or viral genomes, are in part complementary to those at the exon-intron and intron-exon joint sites of ‘pre-mRNA’ [2, 32, 361. Finally, the molecular behaviour of LnRNA appears to be subject to alterations in the physiological and pathological conditions of cells [2, and Patel and Holoubek, in preparation]. Whereas earlier ideas, mainly based on the above findings, suggested that LnRNA promotes splicing by structures formed at the exon-intron or intron-exon joint sites, we suggest in this paper that the association of LnRNA with ‘pre-mRNA’ at the joint sites prevents splicing, which would otherwise occur.

Using a computer program for complementarity assessment of nucleotide sequences [24], we examine in this paper, whether LnRNA, i.e., U1 RNA of rat ascites tumour cells [37] , can potentially be base-paired to the nucleotide sequences involved in the intramolecular double-stranded hairpin structures of rat ‘pre-mRNA‘, and thus whether it is capable of preventing the formation of these hairpin structures.

The result obtained in this assessment is discussed in relation to the regulatory mechanism for RNA processing and message selection during differentia- tion.

3. Intramolecular Double-Stranded Hairpi Structures of Rat ‘he-mRNA’

Of the LnRNA species studied so far, U1 RNA has been fully sequenced [37]. Since this RNA species is of rat tissue origin, the complementarity assessment [24] was carried out between U1 RNA and a rat ‘pre-mRNA’, namely preproinsulin ‘pre-mRNA’. The complete nucleotide sequence of ‘pre-mRNA’ was deduced from that of the rat preproinsulin I1 gene [38], assuming as described previously [24] that both 5‘- and 3’-terminal nucleotide sequences of ‘pre-mRNA’ correspond to those of mRNA (391. As shown in Fig. 1, rat preproinsulin ‘pre-mRNA’ consists of the transcript of three exons (I, 11, and 111) intervened with two introns (IVS 1 and 2). (‘Pre-mRNA’, as opposed to pre-mRNA, means a presumptive mRNA precursor hypothetically tran- scribed from the entire gene of which the nucleotide sequence is known, but does not necessarily imply the pre-mRNA experimentally observed [24].).

In rat preproinsulin ‘pre-mRNA’, complementar- ity assessment showed that intramolecular dou- ble-stranded hairpin structures similar to those hypothetically constructed in chicken ovalbumin, mouse /Pa’ globin, and silk fibroin ‘pre- mRNA’ [24], can be constructed around the 5‘-terminal region of the molecule, with the first exon-intron (I-IVS 1)

Rat Preproinsulin “Pre-mRNA

IVS 1 IVS 2 lntron

I 4 -AAA

I n D Exon

c

0.6 0.8 1.0 (kb) 0 0.2 0.4

Fl i . 1. Arrangement of the transcript of introns and exons of rat preproinsulin ‘pre-mRNA’. In this paper, the terms exons (I, 11, and III), = and introns (IVS 1 and 2), 0, mean the transcripts of corresponding portions of the rat preproinsulin I1 gene [38]. The short arrows pointing to exon I1 and exon 111 show the location of the AUG start triplet and the end of the coding sequence, respectively. A cap structure, CAP, and poly(A) segment, AAA, are shown to indicate the S’-and 3’-ends of the molecules respectively

H. Naora and N. 1. Deacon: RNA Processing Mechanism 127

PorCiin of Possible Binding Sites of Rat Ul RNA to Joint S i i of Rat Preproinsulin ”Pre-mRNA”

c c * u c A A G

A C A A 0 A

G Q : I 5:. u - b

R a t Prepro- 1 $

insulin c:: 1 : ”Pre-mRN&’ 2 -

G - U . A A - U’

c u A C G - C

A G A D u u C - Q C - Q C - G. G - C.‘

:-:

m’0pppA - u.. . --

Fig. 2. The intramolecular and intermolecular structures hypo- thetically constructed at the 5‘-terminal region and around the exon-intron joint site of rat preproinsulin ‘pre-mRNA’. The intramolecular 5’-terminal double-stranded hairpin structure (a) consists of the 5’-terminal region and the nucleotide sequence around the exon-intron (I-IVS 1) joint site. In (b), U1 RNA interacts with the nucleotide sequence around the joint site (I-IVS 1) of ‘pre-mRNA’ and thus prevents the formation of the intramolecular double-stranded hairpin structure (a). Note that the possible binding of U1 RNA takes place at two separate regions around the joint site of ‘pre-mRNA’ and the U1 RNA molecule forms an omega shape. Some known nucleotide sequences are represented by broken line. 0 marks the last nucleotide of exon I. The cap structures are shown only to indicate the S-ends of the molecules

joint site (Fig. 2a). It was also observed that the next intron-exon (IVS 1-11) joint site can also be base-paired with the nucleotides present at the 5’-terminal region, and the nucleotides to be spliced may be positioned next to each other (data not shown). These findings confirm the previous obser- vation made with a few types of ‘pre-mRNA’ ~ 4 1 .

4. Formation of Intermolecular Double-Stranded Structures in Preproinsulin ‘Re-mRNA’ with u1 RNA

Further complementarity assessment showed that preproinsulin ‘pre-mRNA’ , 1061 nucleotides long, possesses 42 regions which are capable of forming stable double-stranded intermolecular structures (with a sufficient number of base-pairs, i.e. , more than six contiguous base-pairs in terms of A-U and G- C base-pairing) with nucleotide sequences of U1 RNA. Fifteen of these regions, (15/42)X 100 = 36%, are located around the four joint sites (I-IVS 1, IVS 1-11, 11-IVS 2 and IVS 2-111; 20, 47, 27, and 54

150 I 0 0 50 1

I S’.Terminur i J’-Terminur

Joint Sihr of ” ?re-mRNA”

Exon I - IVS 1 0 &” I

NS 1 - Exon II ,d ?/A - Exon II - NS 2 0 >a I

IVS 2 - Exon 111 V/A Y %% - YH,

Fig. 3. Location of nucleotide sequences in Ul RNA, capable of forming intermolecular double-stranded structures with nucleotide sequences around the exon-intron and intron-exon joint sites of rat preproinsulin ‘pre-mRNA’. The location of the double-stranded structures, potentially formed with more than six contiguous base-pairs in terms of A-U and G-C base-pairing, are shown in the U1 RNA molecule. The possible binding takes place both at the 5’-terminal region (=) and either around the middle (D) or at the 3’-terminal region (U) to form an omega (a) shape. (In some cases it is possible that the binding also occurs at the 5’-terminal region (=) and around the middle (EZA) to form a broken circle (0) shape). Individual nucleotides are numbered, starting from the 5’-end

nucleotides long respectively) of the molecule (see Note added on proof). If these 42 regions of nucleotide sequences were evenly distributed within the molecule, the four joint sites should correspond to only six regions, that is 42 regions X [(20 + 47 + 27 + 54)/1,061]. Therefore, the distribution of those particular nucleotide sequences within the ‘pre-mRNA’ molecule appears not to be random. The possible binding of U1 RNA took place at two separate regions to form an omega shape (or in some cases, a broken circle shape) around the joint sites of the ‘pre-mRNA’, i.e., one at or near the exon and another at or near the intron (Fig. 2b). This type of possible binding can also be observed in human preproinsulin ‘pre-mRNA’ , hypothetically interacted with adenovirus 2-coded VA (low-molecular-weight) RNAI [40]. The nucleotide sequences of U1 RNA, responsible for base-pairing at the joint sites of rat preproinsulin ‘pre-mRNA’, are confined to restricted regions: one at the 5’-terminal region and another around the middle or at the 3’-terminal region of the U1 RNA molecule (Fig. 3). Using a computer program, we have examined the possible presence of nucleotide sequences in rat Ul RNA, homologous or complementary to the ‘human Alu family consensus’ DNA sequences which are often present in dou- ble-stranded RNA sequences prepared from hetero- geneous nuclear RNA [41-431. The result shows no

128 H. Naora and N. J. Deacon: RNA Processing Mechanism

Table 1. (amended with new nucleotide sequences of UlA RNA) Intermolecular double-stranded structures hypothetically constructed between UI RNA and exon-intron or intron-exon joint sites of preproinsulin 'pre-mRNA'

Model Location Regionsa No. of Stability of base- local structure, pairingsb free energy, AG,

at 2 5 ° C (kcal)

1. Present modeld I-IVS 1 Exon 6 - 13.8 Intron 9 - 15.4

IVS 1-11 Intron 11 - 29.0 Exon 9 - 16.8

11-IVS 2 Exon 6 - 20.4 Intron 10 - 21.6

Ivs 2-111 Intron I - 19.4 Exon 10 - 18.2

2. Model by IVS 1 5'-terminus 6 - 6.0 Lerner et al. 3'-terminus 7 - 9.6

Ivs 2 5'-terminus 5 - 1.4 3'-terminus 9 - 18.8

13-21

a The binding hypothetically takes place at two separate regions in both the model described in this paper and that proposed by Lerner et al. [32]: one at or near the exon and another at or near the intron of the 'pre-mRNA' molecule according to our model, and one at the 5'- and another at the 3'-terminal regions of the intron according to the latter model. Local structures constructed at the regions are considered separately

Base-pairing potentially occurring in the stable double-stranded structure are counted The thermodynamic stabilities of individual structures are calculated according to Tin- et al.

The structures constructed with nucleotide sequences marked by and U of U1 RNA (Fig. 3) are 1441

listed

Possible Regulatory Mechanisms of RNA Splicing "Pro-mRNA" -. . . . . . . . .............................. DO00

Regulatory RNA ' I / **noa

Formation 01 proper . . . . .. hoirpin structurms i : ..... Association of

Regulatory RNA i : Inhibition of formation

Q...... q 000" i hoirpin structures .... .- - *. '. . . .$* *' ,v 0,10" 4.. .. . \. Fig. 4. Schematic representation of possible regulatory mechanism of RNA splicing. When intramolecular double-stranded hairpin structures are formed in the 'pre-mRNA' molecule, the molecule is properly processed for mature mRNA (a). However, if the formation of the haimin structures is inhibited bv association of

Proper processing

*'a" . 1 . . . . . . . 1 Improper processing

011 ? . . .. .. )""

8 ' '""= ... so

z-3 Mature mRNA 22.. 8:

1 No mature mRNA

(bl

a re'gulatory RNA (LnRNA) a ione or more joint sites, no mature mRNA is produced (b).

, exon; 0 0 0, intron; 0 0 0, poly (A) segment

close homology between them (only 62% homology) compared with that between human Alu family consensus DNA sequences and LnRNA from Chi- nese hamster ovary cells (> 90% homology [43]).

ble-stranded structures hypothetically constructed at

the joint sites are thermodynamically stable and hence are capable of preventing formation of the intramolecular 5'- and 3'-terminal double-stranded hairpin structures (see Fig. 2b). It seems likely that

Table 1 shows that the intermolecular dou- possible association of U1 RNA not only to the first exon-intron (I-IVS 1) joint site, but also to one or

H. Naora and N. J. Deacon: RNA Processing Mechanism

some other joint sites still prevents the formation of the proper intramolecular double-stranded structures and eventually interrupts splicing of the exons (Fig. 4). This may imply a regulatory (inhibitory) role for U1 RNA in the processing of preproinsulin ‘pre-mRNA’ .

129

5. Discussion

No complete nucleotide sequence of rat genes, other than that for preproinsulin, is at present available for complementarity assessment. Nucleotide sequences in the immediate vicinity of the joint sites of various types of ‘pre-mRNA’ are not markedly different from each other [32, 45, 461. This would suggest that U1 RNA may well play a regulatory role in the splicing of other types of ‘pre-mRNA’, which contain many introns, in a manner similar to that suggested for preproinsulin ‘pre-mRNA’. However, this does not necessarily exclude the possibility that the wider range interaction between LnRNA and ‘pre-mRNA’ around the joint sites might be involved in the selection of different ‘pre-mRNA’ molecules for processing (see below).

Recently, Lerner et al. [32] suggested an alter- native form of the intermolecular binding of U1 RNA to the ‘pre-mRNA’ molecule, i.e., at both terminal nucleotide sequences of an intron in such a way as to align two joint sites, and proposed a template function of Ul RNA for the correct splicing of ‘pre-mRNA’. A similar model has been proposed in the adenovirus 2 system by Murray and Holliday [36]. When the * hypothetically constructed structures of Lerner et al. [32] are compared with those described here, there are fewer base-pairings involved in the former, and the local structures are thermodynami- cally less stable (Table 1). Our model of RNA splicing indicates that a ‘pre-mRNA’ molecule pos- sesses its own splicing mechanism within the molecule and that the splicing does not require the intermo- lecular aid of RNA [24]. In this paper, particular emphasis is placed on the possibility of the regulatory (inhibitory) role of Ul RNA in RNA splicing, although the possibility raised by the above two research groups cannot be excluded at present.

A striking result recently obtained in comparative studies of sequence complexity and the particular message sequences between nuclear and cytoplasmic RNA of various cell types is that only a few transcribed RNA species are properly processed, transported and translated in cytoplasm [2-121. For example, virtually all of the message sequences

required for sea urchin embryos at the blastula stage are transcribed and present in adult cell nuclei, together with the message sequences needed for adult cells. However, the former sequences are absent in the cytoplasm of adult cells, although the latter sequences are selectively processed and transported into the cytoplasm [lo]. The direct implication of the regulatory role of LnRNA in RNA splicing proposed in this paper would be that the type and even the amount of cytoplasmic mRNA are controlled in such a way that LnRNA specifically binds to the ‘pre-mRNA’ molecules which are transcribed and not required to be processed to cytoplasmic mRNA, resulting in an immediate halt of RNA splicing and/or in abortive processing of the unrequired ‘pre-mRNA’. However, the nature of the mechanism by which the ‘pre-mRNA’ molecules required for proper processing are specifically selected from others st i l l remains to be investigated. There are several possible ways in which this could be achieved, for example: the existence of differentiation-specific LnRNAs, though not highly likely, and/or an alter- ation in RNA-protein interaction including splicing enzymes and HnRNP. The configuration of the pre-mRNA molecule in HnRNP may in part be responsible for such selection. However, it is as yet premature to speculate on the details of the mech- anism. These details may be elucidated by studying the actual structures and nucleotide sequences involved, together with their specific role in the processing of pre-mRNA.

The proposed model can be experimentally examined in various ways. For example, comparative characterisation bepeen those intermolecular dou- ble-stranded structures, if formed with LnRNA in situ, in globin pre-mRNA transcribed in nonerythro- poietic cells [ 5 , 8 ] , and those formed in erythropoietic cells, will provide some general information on the message sequence selection mechanism operating during differentiation of cell functions in relation to the model presented here.

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Note Added in Proof Since this paper was submitted, a few papers have been brought to our attention. Branlant et al. [47] have recently reexamined the nucleotide sequence of rat brain UlA RNA, using both enzymic and chemical treatments of end-labelled RNA, and have found it to differ at several sites from that previously reported (371.

A few alterations must therefore be made: (1) The prepro- insulin “pre-mRNA possesses 43 regions (instead of 42 regions) which are capable of forming stable double-stranded intermolec- ular structures with UIA RNA. Thirty percent of them are located around the joint sites of the “pre-mRNA”. (2) Only a few positions of the possible binding sites at the middle or 3’-terminal regions (shown in Fig. 3) of UIA RNA to the joint sites are to be altered. These alterations in no way aflecr the described model. In fact, the amended nucleotide sequence has made more marked rhe difference between the hypothetically constructed structures of Lerner et al. [32] and our own (see amended Table).

There is about one molecule (on average) of LnRNA per 2500 nucleotides of HnRNA in HnRNP [48,49], but approximately half of these LnRNA are not directly bound to HnRNA. It seems likely therefore that there are two types of HnRNA in HnRNP, one with LnRNA directly bound, and the other free of LnRNA. It may be, therefore, that the former type of HnRNA represents molecules which are not required to be processed to mature mRNA, and the latter type represents molecules which are to be 90 processed.

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48. Gallinaro H, Jacob M (1981) The status of small nuclear RNA in the ribonucleoprotein fibrils containing heterogeneous nuclear RNA. Biochim Biophys Acta 652: 109

49. Gallinaro H, Jacob M (1979) An evaluation of small nuclear RNA in hnRNP. FEBS Lett 104: 176

Received August 1980/Accepted January 1981